Compositions and methods related to protein a (spa) variants

ABSTRACT

Disclosed are methods and compositions for treating or preventing a  Staphylococcus  bacterial infection using a non-toxigenic Protein A (SpA) variant.

This application claims benefit of priority to U.S. ProvisionalApplication Ser. No. 61/166,432, filed Apr. 3, 2009, U.S. ProvisionalApplication Ser. No. 61/237,956, filed Aug. 28, 2009, and U.S.Provisional Application Ser. No. 61/287,996, filed Dec. 18, 2009, theentire contents of all are hereby incorporated by reference.

This invention was made with government support under AI057153, AI75258,AI052474, and GM007281 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

I. Field of the Invention

The present invention relates generally to the fields of immunology,microbiology, and pathology. More particularly, it concerns methods andcompositions involving bacterial Protein A variants, which can be usedto invoke an immune response against the bacteria.

II. Background

The number of both community acquired and hospital acquired infectionshave increased over recent years with the increased use of intravasculardevices. Hospital acquired (nosocomial) infections are a major cause ofmorbidity and mortality, more particularly in the United States, whereit affects more than 2 million patients annually. The most frequentinfections are urinary tract infections (33% of the infections),followed by pneumonia (15.5%), surgical site infections (14.8%) andprimary bloodstream infections (13%) (Emorl and Gaynes, 1993).

The major nosocomial pathogens include Staphylococcus aureus,coagulase-negative Staphylococci (mostly Staphylococcus epidermidis),enterococcus spp., Escherichia coli and Pseudomonas aeruginosa. Althoughthese pathogens cause approximately the same number of infections, theseverity of the disorders they can produce combined with the frequencyof antibiotic resistant isolates balance this ranking towards S. aureusand S. epidermidis as being the most significant nosocomial pathogens.

Staphylococci can cause a wide variety of diseases in humans and otheranimals through either toxin production or invasion. Staphylococcaltoxins are also a common cause of food poisoning, as the bacteria cangrow in improperly-stored food.

Staphylococcus epidermidis is a normal skin commensal which is also animportant opportunistic pathogen responsible for infections of impairedmedical devices and infections at sites of surgery. Medical devicesinfected by S. epidermidis include cardiac pacemakers, cerebrospinalfluid shunts, continuous ambulatory peritoneal dialysis catheters,orthopedic devices and prosthetic heart valves.

Staphylococcus aureus is the most common cause of nosocomial infectionswith a significant morbidity and mortality. It is the cause of somecases of osteomyelitis, endocarditis, septic arthritis, pneumonia,abscesses, and toxic shock syndrome. S. aureus can survive on drysurfaces, increasing the chance of transmission. Any S. aureus infectioncan cause the staphylococcal scalded skin syndrome, a cutaneous reactionto exotoxin absorbed into the bloodstream. It can also cause a type ofsepticemia called pyaemia that can be life-threatening. Problematically,Methicillin-resistant Staphylococcus aureus (MRSA) has become a majorcause of hospital-acquired infections.

S. aureus and S. epidermidis infections are typically treated withantibiotics, with penicillin being the drug of choice, whereasvancomycin is used for methicillin resistant isolates. The percentage ofstaphylococcal strains exhibiting wide-spectrum resistance toantibiotics has become increasingly prevalent, posing a threat foreffective antimicrobial therapy. In addition, the recent emergence ofvancomycin resistant S. aureus strain has aroused fear that MRSA strainsare emerging and spreading for which no effective therapy is available.

An alternative to antibiotic treatment for staphylococcal infections isunder investigation that uses antibodies directed against staphylococcalantigens. This therapy involves administration of polyclonal antisera(WO00/15238, WO00/12132) or treatment with monoclonal antibodies againstlipoteichoic acid (WO98/57994).

An alternative approach would be the use of active vaccination togenerate an immune response against staphylococci. The S. aureus genomehas been sequenced and many of the coding sequences have been identified(WO02/094868, EP0786519), which can lead to the identification ofpotential antigens. The same is true for S. epidermidis (WO01/34809). Asa refinement of this approach, others have identified proteins that arerecognized by hyperimmune sera from patients who have sufferedstaphylococcal infection (WO01/98499, WO02/059148).

S. aureus secretes a plethora of virulence factors into theextracellular milieu (Archer, 1998; Dinges et al., 2000; Foster, 2005;Shaw et al., 2004; Sibbald et al., 2006). Like most secreted proteins,these virulence factors are translocated by the Sec machinery across theplasma membrane. Proteins secreted by the Sec machinery bear anN-terminal leader peptide that is removed by leader peptidase once thepre-protein is engaged in the Sec translocon (Dalbey and Wickner, 1985;van Wely et al., 2001). Recent genome analysis suggests thatActinobacteria and members of the Firmicutes encode an additionalsecretion system that recognizes a subset of proteins in aSec-independent manner (Pallen, 2002). ESAT-6 (early secreted antigentarget 6 kDa) and CFP-10 (culture filtrate antigen 10 kDa) ofMycobacterium tuberculosis represent the first substrates of this novelsecretion system termed ESX-1 or 5 nm in M. tuberculosis (Andersen etal., 1995; Hsu et al., 2003; Pym et al., 2003; Stanley et al., 2003). InS. aureus, two ESAT-6 like factors designated EsxA and EsxB are secretedby the Ess pathway (ESAT-6 secretion system) (Burts et al., 2005).

The first generation of vaccines targeted against S. aureus or againstthe exoproteins it produces have met with limited success (Lee, 1996).There remains a need to develop effective vaccines againststaphylococcal infections. Additional compositions for treatingstaphylococcal infections are also needed.

SUMMARY OF THE INVENTION

Protein A (SpA) (SEQ ID NO:33), a cell wall anchored surface protein ofStaphylococcus aureus, provides for bacterial evasion from innate andadaptive immune responses. Protein A binds immunoglobulins at their Fcportion, interacts with the VH3 domain of B cell receptorsinappropriately stimulating B cell proliferation and apotosis, binds tovon Willebrand factor A1 domains to activate intracellular clotting, andalso binds to the TNF Receptor-1 to contribute to the pathogenesis ofstaphylococcal pneumonia. Due to the fact that Protein A capturesimmunoglobulin and displays toxic attributes, the possibility that thissurface molecule may function as a vaccine in humans has not beenrigorously pursued. Here the inventors demonstrate that Protein Avariants no longer able to bind to immunoglobulins, which are therebyremoved of their toxigenic potential, i.e., are non-toxigenic, stimulatehumoral immune responses that protect against staphylococcal disease.

In certain embodiments the SpA variant is a full length SpA variantcomprising a variant A, B, C, D, and E domain. In certain aspects, theSpA variant comprises or consists of the amino acid sequence that is 80,90, 95, 98, 99, or 100% identical to the amino acid sequence of SEQ IDNO:34 In other embodiments the SpA variant comprises a segment of SpA.The SpA segment can comprise at least or at most 1, 2, 3, 4, 5 or moreIgG binding domains. The IgG domains can be at least or at most 1, 2, 3,4, 5 or more variant A, B, C, D, or E domains. In certain aspects theSpA variant comprises at least or at most 1, 2, 3, 4, 5, or more variantA domains. In a further aspect the SpA variant comprises at least or atmost 1, 2, 3, 4, 5, or more variant B domains. In still a further aspectthe SpA variant comprises at least or at most 1, 2, 3, 4, 5, or morevariant C domains. In yet a further aspect the SpA variant comprises atleast or at most 1, 2, 3, 4, 5, or more variant D domains. In certainaspects the SpA variant comprises at least or at most 1, 2, 3, 4, 5, ormore variant E domains. In a further aspect the SpA variant comprises acombination of A, B, C, D, and E domains in various combinations andpermutations. The combinations can include all or part of a SpA signalpeptide segment, a SpA region X segment, and/or a SpA sorting signalsegment. In other aspects the SpA variant does not include a SpA signalpeptide segment, a SpA region X segment, and/or a SpA sorting signalsegment. In certain aspects a variant A domain comprises a substitutionat position(s) 7, 8, 34, and/or 35 of SEQ ID NO:4. In another aspect avariant B domain comprises a substitution at position(s) 7, 8, 34,and/or 35 of SEQ ID NO:6. In still another aspect a variant C domaincomprises a substitution at position(s) 7, 8, 34, and/or 35 of SEQ IDNO:5. In certain aspects a variant D domain comprises a substitution atposition(s) 9, 10, 37, and/or 38 of SEQ ID NO:2. In a further aspect avariant E domain comprises a substitution at position(s) 6, 7, 33,and/or 34 of SEQ ID NO:3.

In certain aspects the SpA variant includes a substitution of (a) one ormore amino acid substitution in an IgG Fc binding sub-domain of SpAdomain A, B, C, D, and/or E that disrupts or decreases binding to IgGFc, and (b) one or more amino acid substitution in a V_(H)3 bindingsub-domain of SpA domain A, B, C, D, and/or E that disrupts or decreasesbinding to V_(H)3. In still further aspects the amino acid sequence of aSpA variant comprises an amino acid sequence that is at least 50%, 60%,70%, 80%, 90%, 95%, or 100% identical, including all values and rangesthere between, to the amino acid sequence of SEQ ID NOs:2-6.

In a further aspect the SpA variant includes (a) one or more amino acidsubstitution in an IgG Fc binding sub-domain of SpA domain D, or at acorresponding amino acid position in other IgG domains, that disrupts ordecreases binding to IgG Fc, and (b) one or more amino acid substitutionin a V_(H)3 binding sub-domain of SpA domain D, or at a correspondingamino acid position in other IgG domains, that disrupts or decreasesbinding to V_(H)3. In certain aspects amino acid residue F5, Q9, Q10,S11, F13, Y14, L17, N28, 131, and/or K₃₅ (SEQ ID NO:2,QQNNFNKDQQSAFYEILNMPNLNEAQRNGFIQSLKDDPSQSTNVLGEAKKLN ES) of the IgG Fcbinding sub-domain of domain D are modified or substituted. In certainaspects amino acid residue Q26, G29, F30, S33, D36, D37, Q40, N43,and/or E47 (SEQ ID NO:2) of the V_(H)3 binding sub-domain of domain Dare modified or substituted such that binding to Fc or V_(H)3 isattenuated. In further aspects corresponding modifications orsubstitutions can be engineered in corresponding positions of the domainA, B, C, and/or E. Corresponding positions are defined by alignment ofthe domain D amino acid sequence with one or more of the amino acidsequences from other IgG binding domains of SpA, for example see FIG. 1.In certain aspects the amino acid substitution can be any of the other20 amino acids. In a further aspect conservative amino acidsubstitutions can be specifically excluded from possible amino acidsubstitutions. In other aspects only non-conservative substitutions areincluded. In any event, any substitution or combination of substitutionsthat reduces the binding of the domain such that SpA toxicity issignificantly reduced is contemplated. The significance of the reductionin binding refers to a variant that produces minimal to no toxicity whenintroduced into a subject and can be assessed using in vitro methodsdescribed herein.

In certain embodiments, a variant SpA comprises at least or at most 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more variant SpA domain D peptides. Incertain aspects 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, or 19 or more amino acid residues of the variant SpA aresubstituted or modified—including but not limited to amino acids F5, Q9,Q10, S11, F13, Y14, L17, N28, I31, and/or K35 (SEQ ID NO:2) of the IgGFc binding sub-domain of domain D and amino acid residue Q26, G29, F30,S33, D36, D37, Q40, N43, and/or E47 (SEQ ID NO:2) of the V_(H)3 bindingsub-domain of domain D. In one aspect of the invention glutamineresidues at position 9 and/or 10 of SEQ ID NO:2 (or correspondingpositions in other domains) are mutated. In another aspect, asparticacid residues 36 and/or 37 of SEQ ID NO:2 (or corresponding positions inother domains) are mutated. In a further aspect, glutamine 9 and 10, andaspartic acid residues 36 and 37 are mutated. Purified non-toxigenic SpAor SpA-D mutants/variants described herein are no longer able tosignificantly bind (i.e., demonstrate attenuated or disrupted bindingaffinity) Fcγ or F(ab)₂ V_(H)3 and also do not stimulate B cellapoptosis. These non-toxigenic Protein A variants can be used as subunitvaccines and raise humoral immune responses and confer protectiveimmunity against S. aureus challenge. Compared to wild-type full-lengthProtein A or the wild-type SpA-domain D, immunization with SpA-Dvariants resulted in an increase in Protein A specific antibody. Using amouse model of staphylococcal challenge and abscess formation, it wasobserved that immunization with the non-toxigenic Protein A variantsgenerated significant protection from staphylococcal infection andabscess formation. As virtually all S. aureus strains express Protein A,immunization of humans with the non-toxigenic Protein A variants canneutralize this virulence factor and thereby establish protectiveimmunity. In certain aspects the protective immunity protects orameliorates infection by drug resistant strains of Staphylococcus, suchas USA300 and other MRSA strains.

Embodiments include the use of Protein A variants in methods andcompositions for the treatment of bacterial and/or staphylococcalinfection. This application also provides an immunogenic compositioncomprising a Protein A variant or immunogenic fragment thereof. Incertain aspects, the immunogenic fragment is a Protein A domain Dsegment. Furthermore, the present invention provides methods andcompositions that can be used to treat (e.g., limiting staphylococcalabscess formation and/or persistence in a subject) or prevent bacterialinfection. In some cases, methods for stimulating an immune responseinvolve administering to the subject an effective amount of acomposition including or encoding all or part of a Protein A variantpolypeptide or antigen, and in certain aspects other bacterial proteins.Other bacterial proteins include, but are not limited to (i) a secretedvirulence factor, and/or a cell surface protein or peptide, or (ii) arecombinant nucleic acid molecule encoding a secreted virulence factor,and/or a cell surface protein or peptide.

In other aspects, the subject can be administered all or part of aProtein A variant, such as a variant Protein A domain D segment. Thepolypeptide of the invention can be formulated in a pharmaceuticallyacceptable composition. The composition can further comprise one or moreof at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, or 19 additional staphylococcal antigen or immunogenicfragment thereof (e.g., Eap, Ebh, Emp, EsaB, EsaC, EsxA, EsxB, SdrC,SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, Coa, Hla (e.g., H35 mutants), IsdC,SasF, vWbp, or vWh). Additional staphylococcal antigens that can be usedin combination with a Protein A variant include, but are not limited to52 kDa vitronectin binding protein (WO 01/60852), Aaa (GenBankCAC80837), Aap (GenBank accession AJ249487), Ant (GenBank accessionNP_(—)372518), autolysin glucosaminidase, autolysin amidase, Cna,collagen binding protein (U.S. Pat. No. 6,288,214), EFB (FIB), Elastinbinding protein (EbpS), EPB, FbpA, fibrinogen binding protein (U.S. Pat.No. 6,008,341), Fibronectin binding protein (U.S. Pat. No. 5,840,846),FnbA, FnbB, GehD (US 2002/0169288), HarA, HBP, Immunodominant ABCtransporter, IsaA/P isA, laminin receptor, Lipase GehD, MAP, Mg2+transporter, MHC II analogue (U.S. Pat. No. 5,648,240), MRPII, Npase,RNA III activating protein (RAP), SasA, SasB, SasC, SasD, SasK,SBI, SdrF(WO 00/12689), SdrG/Fig (WO 00/12689), SdrH (WO 00/12689), SEA exotoxins(WO 00/02523), SEB exotoxins (WO 00/02523), SitC and Ni ABC transporter,SitC/MntC/saliva binding protein (U.S. Pat. No. 5,801,234), SsaA, SSP-1,SSP-2, and/or Vitronectin binding protein (see PCT publicationsWO2007/113222, WO2007/113223, WO2006/032472, WO2006/032475,WO2006/032500, each of which is incorporated herein by reference intheir entirety). The staphylococcal antigen or immunogenic fragment canbe administered concurrently with the Protein A variant. Thestaphylococcal antigen or immunogenic fragment and the Protein A variantcan be administered in the same composition. The Protein A variant canalso be a recombinant nucleic acid molecule encoding a Protein Avariant. A recombinant nucleic acid molecule can encode the Protein Avariant and at least one staphylococcal antigen or immunogenic fragmentthereof. As used herein, the term “modulate” or “modulation” encompassesthe meanings of the words “enhance,” or “inhibit.” “Modulation” ofactivity may be either an increase or a decrease in activity. As usedherein, the term “modulator” refers to compounds that effect thefunction of a moiety, including up-regulation, induction, stimulation,potentiation, inhibition, down-regulation, or suppression of a protein,nucleic acid, gene, organism or the like.

In certain embodiments the methods and compositions use or include orencode all or part of the Protein A variant or antigen. In otheraspects, the Protein A variant may be used in combination with secretedfactors or surface antigens including, but not limited to one or more ofan isolated Eap, Ebh, Emp, EsaB, EsaC, EsxA, EsxB, SdrC, SdrD, SdrE,IsdA, IsdB, ClfA, ClfB, Coa, Hla, IsdC, SasF, vWbp, or vWh polypeptideor immunogenic segment thereof. Additional staphylococcal antigens thatcan be used in combination with a Protein A variant include, but are notlimited to 52 kDa vitronectin binding protein (WO 01/60852), Aaa, Aap,Ant, autolysin glucosaminidase, autolysin amidase, Cna, collagen bindingprotein (U.S. Pat. No. 6,288,214), EFB (FIB), Elastin binding protein(EbpS), EPB, FbpA, fibrinogen binding protein (U.S. Pat. No. 6,008,341),Fibronectin binding protein (U.S. Pat. No. 5,840,846), FnbA, FnbB, GehD(US 2002/0169288), HarA, HBP, Immunodominant ABC transporter, IsaA/PisA, laminin receptor, Lipase GehD, MAP, Mg2+ transporter, MHC IIanalogue (U.S. Pat. No. 5,648,240), MRPII, Npase, RNA III activatingprotein (RAP), SasA, SasB, SasC, SasD, SasK,SBI, SdrF (WO 00/12689),SdrG/Fig (WO 00/12689), SdrH (WO 00/12689), SEA exotoxins (WO 00/02523),SEB exotoxins (WO 00/02523), SitC and Ni ABC transporter,SitC/MntC/saliva binding protein (U.S. Pat. No. 5,801,234), SsaA, SSP-1,SSP-2, and/or Vitronectin binding protein. In certain embodiments, 1, 2,3, 4, 5, 6, 7, 8, 9, 10 or more of Eap, Ebh, Emp, EsaB, EsaC, EsxA,EsxB, SdrC, SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, Coa, Hla, IsdC, SasF,vWbp, vWh, 52 kDa vitronectin binding protein (WO 01/60852), Aaa, Aap,Ant, autolysin glucosaminidase, autolysin amidase, Cna, collagen bindingprotein (US6288214), EFB (FIB), Elastin binding protein (EbpS), EPB,FbpA, fibrinogen binding protein (U.S. Pat. No. 6,008,341), Fibronectinbinding protein (U.S. Pat. No. 5,840,846), FnbA, FnbB, GehD (US2002/0169288), HarA, HBP, Immunodominant ABC transporter, IsaA/P isA,laminin receptor, Lipase GehD, MAP, Mg2+ transporter, MHC II analogue(U.S. Pat. No. 5,648,240), MRPII, Npase, RNA III activating protein(RAP), SasA, SasB, SasC, SasD, SasK,SBI, SdrF (WO 00/12689), SdrG/Fig(WO 00/12689), SdrH (WO 00/12689), SEA exotoxins (WO 00/02523), SEBexotoxins (WO 00/02523), SitC and Ni ABC transporter, SitC/MntC/salivabinding protein (U.S. Pat. No. 5,801,234), SsaA, SSP-1, SSP-2, and/orVitronectin binding protein. can be specifically excluded from aformulation of the invention.

In still further aspects, the isolated Protein A variant ismultimerized, e.g., dimerized or a linear fusion of two or morepolypeptides or peptide segments. In certain aspects of the invention, acomposition comprises multimers or concatamers of 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more isolated cellsurface proteins or segments thereof. Concatamers are linearpolypeptides having one or more repeating peptide units. SpApolypeptides or fragments can be consecutive or separated by a spacer orother peptide sequences, e.g., one or more additional bacterial peptide.In a further aspect, the other polypeptides or peptides contained in themultimer or concatamer can include, but are not limited to 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 of Eap, Ebh, Emp,EsaB, EsaC, EsxA, EsxB, SdrC, SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, Coa,Hla, IsdC, SasF, vWbp, vWh or immunogenic fragments thereof. Additionalstaphylococcal antigens that can be used in combination with a Protein Avariant include, but are not limited to 52 kDa vitronectin bindingprotein (WO 01/60852), Aaa, Aap, Ant, autolysin glucosaminidase,autolysin amidase, Cna, collagen binding protein (U56288214), EFB (FIB),Elastin binding protein (EbpS), EPB, FbpA, fibrinogen binding protein(U.S. Pat. No. 6,008,341), Fibronectin binding protein (U.S. Pat. No.5,840,846), FnbA, FnbB, GehD (US 2002/0169288), HarA, HBP,Immunodominant ABC transporter, IsaA/P isA, laminin receptor, LipaseGehD, MAP, Mg2+ transporter, MHC II analogue (U.S. Pat. No. 5,648,240),MRPII, Npase, RNA III activating protein (RAP), SasA, SasB, SasC, SasD,SasK,SBI, SdrF (WO 00/12689), SdrG/Fig (WO 00/12689), SdrH (WO00/12689), SEA exotoxins (WO 00/02523), SEB exotoxins (WO 00/02523),SitC and Ni ABC transporter, SitC/MntC/saliva binding protein (U.S. Pat.No. 5,801,234), SsaA, SSP-1, SSP-2, and/or Vitronectin binding protein.

The term “Protein A variant” or “SpA variant” refers to polypeptidesthat include a SpA IgG domain having two or more amino acidsubstitutions that disrupt binding to Fc and V_(H)3. In certain aspect,a SpA variant includes a variant domain D peptide, as well as variantsof SpA polypeptides and segments thereof that are non-toxigenic andstimulate an immune response against staphylococcus bacteria Protein Aand/or bacteria expressing such.

Embodiments of the present invention include methods for eliciting animmune response against a staphylococcus bacterium or staphylococci in asubject comprising providing to the subject an effective amount of aProtein A variant or a segment thereof. In certain aspects, the methodsfor eliciting an immune response against a staphylococcus bacterium orstaphylococci in a subject comprising providing to the subject aneffective amount of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19 or more secreted proteins and/or cell surface proteins orsegments/fragments thereof. A secreted protein or cell surface proteinincludes, but is not limited to Eap, Ebh, Emp, EsaB, EsaC, EsxA, EsxB,SdrC, SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, Coa, Hla, IsdC, SasF, vWbp,and/or vWh proteins and immunogenic fragments thereof. Additionalstaphylococcal antigens that can be used in combination with a Protein Avariant include, but are not limited to 52 kDa vitronectin bindingprotein (WO 01/60852), Aaa, Aap, Ant, autolysin glucosaminidase,autolysin amidase, Cna, collagen binding protein (U.S. Pat. No.6,288,214), EFB (FIB), Elastin binding protein (EbpS), EPB, FbpA,fibrinogen binding protein (U.S. Pat. No. 6,008,341), Fibronectinbinding protein (U.S. Pat. No. 5,840,846), FnbA, FnbB, GehD (US2002/0169288), HarA, HBP, Immunodominant ABC transporter, IsaA/P isA,laminin receptor, Lipase GehD, MAP, Mg2+ transporter, MHC II analogue(U.S. Pat. No. 5,648,240), MRPII, Npase, RNA III activating protein(RAP), SasA, SasB, SasC, SasD, SasK,SBI, SdrF (WO 00/12689), SdrG/Fig(WO 00/12689), SdrH (WO 00/12689), SEA exotoxins (WO 00/02523), SEBexotoxins (WO 00/02523), SitC and Ni ABC transporter, SitC/MntC/salivabinding protein (U.S. Pat. No. 5,801,234), SsaA, SSP-1, SSP-2, and/orVitronectin binding protein.

Embodiments of the invention include compositions that include apolypeptide, peptide, or protein that is or is at least 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% identical or similar to Protein A,or a second protein or peptide that is a secreted bacterial protein or abacterial cell surface protein. In a further embodiment of the inventiona composition may include a polypeptide, peptide, or protein that is oris at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical or similar to a Protein A domain D polypeptide (SEQ ID NO:2),domain E (SEQ ID NO:3), domain A (SEQ ID NO:4), domain C (SEQ ID NO:5),domain B (SEQ ID NO:6), or a nucleic acid sequence encoding a Protein Adomain D, domain E, domain A, domain C, or domain B polypeptide. Incertain aspects a Protein A polypeptide segment will have an amino acidsequence of SEQ ID NO:8. Similarity or identity, with identity beingpreferred, is known in the art and a number of different programs can beused to identify whether a protein (or nucleic acid) has sequenceidentity or similarity to a known sequence. Sequence identity and/orsimilarity is determined using standard techniques known in the art,including, but not limited to, the local sequence identity algorithm ofSmith & Waterman (1981), by the sequence identity alignment algorithm ofNeedleman & Wunsch (1970), by the search for similarity method ofPearson & Lipman (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Drive, Madison,Wis.), the Best Fit sequence program described by Devereux et al.(1984), preferably using the default settings, or by inspection.Preferably, percent identity is calculated by using alignment toolsknown to and readily ascertainable to those of skill in the art. Percentidentity is essentially the number of identical amino acids divided bythe total number of amino acids compared times one hundred.

Still further embodiments include methods for stimulating in a subject aprotective or therapeutic immune response against a staphylococcusbacterium comprising administering to the subject an effective amount ofa composition including (i) a SpA variant, e.g., a variant SpA domain Dpolypeptide or peptide thereof; or, (ii) a nucleic acid moleculeencoding such a SpA variant polypeptide or peptide thereof, or (iii)administering a SpA variant domain D polypeptide with any combination orpermutation of bacterial proteins described herein. In a preferredembodiment the composition is not a staphylococcus bacterium. In certainaspects the subject is a human or a cow. In a further aspect thecomposition is formulated in a pharmaceutically acceptable formulation.The staphylococci may be Staphylococcus aureus.

Yet still further embodiments include vaccines comprising apharmaceutically acceptable composition having an isolated SpA variantpolypeptide, or any other combination or permutation of protein(s) orpeptide(s) described herein, wherein the composition is capable ofstimulating an immune response against a staphylococcus bacterium. Thevaccine may comprise an isolated SpA variant polypeptide, or any othercombination or permutation of protein(s) or peptide(s) described. Incertain aspects of the invention the isolated SpA variant polypeptide,or any other combination or permutation of protein(s) or peptide(s)described are multimerized, e.g., dimerized or concatamerized. In afurther aspect, the vaccine composition is contaminated by less thanabout 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.25, 0.05% (or any rangederivable therein) of other Staphylococcal proteins. A composition mayfurther comprise an isolated non-SpA polypeptide. Typically the vaccinecomprises an adjuvant. In certain aspects a protein or peptide of theinvention is linked (covalently or non-covalently) to the adjuvant,preferably the adjuvant is chemically conjugated to the protein.

In still yet further embodiments, a vaccine composition is apharmaceutically acceptable composition having a recombinant nucleicacid encoding all or part of a SpA variant polypeptide, or any othercombination or permutation of protein(s) or peptide(s) described herein,wherein the composition is capable of stimulating an immune responseagainst a staphylococcus bacteria. The vaccine composition may comprisea recombinant nucleic acid encoding all or part of a SpA variantpolypeptide, or any other combination or permutation of protein(s) orpeptide(s) described herein. In certain embodiments the recombinantnucleic acid contains a heterologous promoter. Preferably therecombinant nucleic acid is a vector. More preferably the vector is aplasmid or a viral vector. In some aspects the vaccine includes arecombinant, non-staphylococcus bacterium containing the nucleic acid.The recombinant non-staphylococci may be Salmonella or anothergram-positive bacteria. The vaccine may comprise a pharmaceuticallyacceptable excipient, more preferably an adjuvant.

Still further embodiments include methods for stimulating in a subject aprotective or therapeutic immune response against a staphylococcusbacterium comprising administering to the subject an effective amount ofa composition of a SpA variant polypeptide or segment/fragment thereofand further comprising one or more of a Eap, Ebh, Emp, EsaB, EsaC, EsxA,EsxB, SdrC, SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, Coa, Hla, IsdC, SasF,vWbp, or vWh protein or peptide thereof. In a preferred embodiment thecomposition comprises a non-staphylococcus bacterium. In a furtheraspect the composition is formulated in a pharmaceutically acceptableformulation. The staphylococci for which a subject is being treated maybe Staphylococcus aureus. Methods of the invention also include SpAvariant compositions that contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19 or more secreted virulence factors and/orcell surface proteins, such as Eap, Ebh, Emp, EsaC, EsxA, EsxB, SdrC,SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, Coa, Hla, IsdC, SasF, vWbp, or vWhin various combinations. In certain aspects a vaccine formulationincludes Eap, Ebh, Emp, EsaC, EsxA, EsxB, SdrC, SdrD, SdrE, IsdA, IsdB,ClfA, ClfB, Coa, Hla, IsdC, SasF, vWbp, and vWh. In certain aspects anantigen combination can include (1) a SpA variant and IsdA; (2) SpAvariant and ClfB; (3) SpA variant and SdrD; (4) SpA variant and Hla orHla variant; (5) SpA variant and ClfB, SdrD, and Hla or Hla variant; (6)SpA variant, IsdA, SdrD, and Hla or Hla variant; (7) SpA variant, IsdA,ClfB, and Hla or Hla variant; (8) SpA variant, IsdA, Clf13, and SdrD;(9) SpA variant, IsdA, ClfB, SdrD and Hla or Hla variant; (10) SpAvariant, IsdA, ClfB, and SdrD; (11) SpA variant, IsdA, SdrD, and Hla orHla variant; (12) SpA variant, IsdA, and Hla or Hla variant; (13) SpAvariant, IsdA, ClfB, and Hla or Hla variant; (14) SpA variant, ClfB, andSdrD; (15) SpA variant, ClfB, and Hla or Hla variant; or (16) SpAvariant, SdrD, and Hla or Hla variant.

In certain aspects, a bacterium delivering a composition of theinvention will be limited or attenuated with respect to prolonged orpersistent growth or abscess formation. In yet a further aspect, SpAvariant(s) can be overexpressed in an attenuated bacterium to furtherenhance or supplement an immune response or vaccine formulation.

Certain embodiments are directed to methods for eliciting an immuneresponse against a staphylococcus bacterium in a subject comprisingproviding to the subject an effective amount of a peptide comprising acoagulase polypeptide or an immunogenic segment thereof having an aminoacid sequence that is at least 80, 85, 90, 95, 98, to 100% identical toSEQ ID NO:27 or a segment thereof or at least 80, 85, 90, 95, 98, to100% identical to amino acids 27-508 of SEQ ID NO:32 or a segmentthereof.

In certain aspects, the subject is provided with an effective amount ofan coagulase polypeptide by administering to the subject a compositioncomprising: (i) an isolated coagulase polypeptide or segment thereofhaving an amino acid sequence that is at least 90% identical to SEQ IDNO:27 or a segment thereof or is at least 90% identical to amino acids27-508 of SEQ ID NO:32 or a segment thereof or (ii) at least oneisolated recombinant nucleic acid molecule encoding a coagulasepolypeptide or a segment thereof having an amino acid sequence that isat least 90% identical to SEQ ID NO:27 or a segment thereof or is atleast 90% identical to amino acids 27-508 of SEQ ID NO:32 or a segmentthereof. In a further aspect, the composition comprises an isolatedcoagulase polypeptide having the amino acid sequence of SEQ ID NO:27 orthe amino acid sequence of amino acids 27-508 of SEQ ID NO:32.

Certain embodiments are directed to methods for treating astaphylococcal infection in a subject comprising providing to a subjecthaving or suspected of having or at risk of developing a staphylococcalinfection an effective amount of an isolated peptide comprising acoagulase polypeptide having an amino acid sequence that is at least 80,85, 90, 95, 98, to 100% identical to SEQ ID NO:27 or is at least 80, 85,90, 95, 98, to 100% identical to amino acids 27-508 of SEQ ID NO:32. Ina particular aspect, the coagulase polypeptide has an amino acidsequence of SEQ ID NO:27 or has an amino acid identical to amino acids27-508 of SEQ ID NO:32. In certain aspects, the subject is diagnosedwith a persistent staphylococcal infection. In a further aspect, thecoagulase polypeptide elicits production of an antibody that binds Coaor vWbpvWh in the subject.

Embodiments include methods of preventing or treating staphylococcalinfection comprising the step of administering an immunogeniccomposition comprising a Staphylococcal coagulase or an immunogenicsegment thereof.

Certain embodiments are directed to methods of preparing animmunoglobulin for use in prevention or treatment of staphylococcalinfection comprising the steps of immunizing a recipient with acoagulase polypeptide and isolating immunoglobulin from the recipient.

A further embodiment is directed to an immunoglobulin prepared by themethod described herein.

A further embodiment is directed to methods for treatment or preventionof staphylococcal infection comprising a step of administering to apatient an effective amount of pharmaceutical preparation ofimmunoglobulin that binds a coagulase.

Other embodiments are directed to a use of the pharmaceuticalpreparation of coagulase immunoglobulins in the manufacture of amedicament for the treatment or prevention of staphylococcal infection.

Yet still further embodiments include vaccines comprising apharmaceutically acceptable composition having an isolated coagulasepolypeptide, or any other combination or permutation of protein(s) orpeptide(s) described herein, wherein the composition is capable ofstimulating an immune response against a staphylococcus bacterium. Thevaccine may comprise an isolated coagulase polypeptide, or any othercombination or permutation of protein(s) or peptide(s) described. Incertain aspects of the invention the isolated coagulase polypeptide, orany other combination or permutation of protein(s) or peptide(s)described are multimerized, e.g., dimerized or concatamerized. In afurther aspect, the vaccine composition is contaminated by less thanabout 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.25, 0.05% (or any rangederivable therein) of other Staphylococcal proteins. A composition mayfurther comprise an isolated non-coagulase polypeptide. Typically thevaccine comprises an adjuvant. In certain aspects a protein or peptideof the invention is linked (covalently or non-covalently) to theadjuvant, preferably the adjuvant is chemically conjugated to theprotein.

In still yet further embodiments, a vaccine composition is apharmaceutically acceptable composition having a recombinant nucleicacid encoding all or part of a coagulase polypeptide, or any othercombination or permutation of protein(s) or peptide(s) described herein,wherein the composition is capable of stimulating an immune responseagainst a staphylococcus bacterium. The vaccine composition may comprisea recombinant nucleic acid encoding all or part of a coagulasepolypeptide, or any other combination or permutation of protein(s) orpeptide(s) described herein. In certain embodiments the recombinantnucleic acid contains a heterologous promoter. Preferably therecombinant nucleic acid is a vector. More preferably the vector is aplasmid or a viral vector. In some aspects the vaccine includes arecombinant, non-staphylococcus bacterium containing the nucleic acid.The recombinant non-staphylococci may be Salmonella or anothergram-positive bacteria. The vaccine may comprise a pharmaceuticallyacceptable excipient, more preferably an adjuvant.

Still further embodiments include methods for stimulating in a subject aprotective or therapeutic immune response against a staphylococcusbacterium comprising administering to the subject an effective amount ofa composition of a coagulase polypeptide or segment/fragment thereof andfurther comprising one or more of a Eap, Ebh, Emp, EsaB, EsaC, EsxA,EsxB, SdrC, SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, Coa, Hla, IsdC, SasF,vWbp, or vWh protein or peptide thereof. In a preferred embodiment thecomposition comprises a non-staphylococcus bacterium. In a furtheraspect the composition is formulated in a pharmaceutically acceptableformulation. The staphylococci for which a subject is being treated maybe Staphylococcus aureus. Methods of the invention also includecoagulase compositions that contain one or more of 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or more secreted virulencefactors and/or cell surface proteins, such as Eap, Ebh, Emp, EsaC, EsxA,EsxB, SdrC, SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, Coa, Hla, IsdC, SasF,SpA and variants thereof, vWbp, or vWh in various combinations. Incertain aspects a vaccine formulation includes Eap, Ebh, Emp, EsaC,EsxA, EsxB, SdrC, SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, Coa, Hla, IsdC,SasF, vWbp, and vWh. In certain aspects an antigen combination caninclude (1) a Coa and/or vWbp, and IsdA; (2) a Coa and/or vWbp, andClfB; (3) a Coa and/or vWbp, and SdrD; (4) a Coa and/or vWbp, and Hla orHla variant; (5) a Coa and/or vWbp, and ClfB, SdrD, and Hla or Hlavariant; (6) a Coa and/or vWbp, and IsdA, SdrD, and Hla or Hla variant;(7) a Coa and/or vWbp, and IsdA, ClfB, and Hla or Hla variant; (8) a Coaand/or vWbp, and IsdA, ClfB, and SdrD; (9) a Coa and/or vWbp, and IsdA,ClfB, SdrD and Hla or Hla variant; (10) a Coa and/or vWbp, and IsdA,ClfB, and SdrD; (11) a Coa and/or vWbp, and IsdA, SdrD, and Hla or Hlavariant; (12) a Coa and/or vWbp, and IsdA, and Hla or Hla variant; (13)a Coa and/or vWbp, and IsdA, ClfB, and Hla or Hla variant; (14) a Coaand/or vWbp, and ClfB, and SdrD; (15) a Coa and/or vWbp, and ClfB, andHla or Hla variant; or (16) a Coa and/or vWbp, and SdrD, and Hla or Hlavariant.

The term “EsxA protein” refers to a protein that includes isolatedwild-type EsxA polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria EsxA proteins.

The term “EsxB protein” refers to a protein that includes isolatedwild-type EsxB polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria EsxB proteins.

The term “SdrD protein” refers to a protein that includes isolatedwild-type SdrD polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria SdrD proteins.

The term “SdrE protein” refers to a protein that includes isolatedwild-type SdrE polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria SdrE proteins.

The term “IsdA protein” refers to a protein that includes isolatedwild-type IsdA polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria IsdA proteins.

The term “IsdB protein” refers to a protein that includes isolatedwild-type IsdB polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria IsdB proteins.

The term “Eap protein” refers to a protein that includes isolatedwild-type Eap polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria Eap proteins.

The term “Ebh protein” refers to a protein that includes isolatedwild-type Ebh polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria Ebh proteins.

The term “Emp protein” refers to a protein that includes isolatedwild-type Emp polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria Emp proteins.

The term “EsaB protein” refers to a protein that includes isolatedwild-type EsaB polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria EsaB proteins.

The term “EsaC protein” refers to a protein that includes isolatedwild-type EsaC polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria EsaC proteins.

The term “SdrC protein” refers to a protein that includes isolatedwild-type SdrC polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria SdrC proteins.

The term “ClfA protein” refers to a protein that includes isolatedwild-type ClfA polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria ClfA proteins.

The term “ClfB protein” refers to a protein that includes isolatedwild-type ClfB polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria ClfB proteins.

The term “Coa protein” refers to a protein that includes isolatedwild-type Coa polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria Coa proteins.

The term “Hla protein” refers to a protein that includes isolatedwild-type Hla polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria Hla proteins.

The term “IsdC protein” refers to a protein that includes isolatedwild-type IsdC polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria IsdC proteins.

The term “SasF protein” refers to a protein that includes isolatedwild-type SasF polypeptides from staphylococcus bacteria and segmentsthereof, as well as variants that stimulate an immune response againststaphylococcus bacteria SasF proteins.

The term “vWbp protein” refers to a protein that includes isolatedwild-type vWbp (von Willebrand factor binding protein) polypeptides fromstaphylococcus bacteria and segments thereof, as well as variants thatstimulate an immune response against staphylococcus bacteria vWbpproteins.

The term “vWh protein” refers to a protein that includes isolatedwild-type vWh (von Willebrand factor binding protein homolog)polypeptides from staphylococcus bacteria and segments thereof, as wellas variants that stimulate an immune response against staphylococcusbacteria vWh proteins.

An immune response refers to a humoral response, a cellular response, orboth a humoral and cellular response in an organism. An immune responsecan be measured by assays that include, but are not limited to, assaysmeasuring the presence or amount of antibodies that specificallyrecognize a protein or cell surface protein, assays measuring T-cellactivation or proliferation, and/or assays that measure modulation interms of activity or expression of one or more cytokines.

In still further embodiments of the invention a composition may includea polypeptide, peptide, or protein that is or is at least 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% identical or similar to an EsxAprotein. In certain aspects the EsxA protein will have all or part ofthe amino acid sequence of SEQ ID NO:11.

In still further embodiments of the invention a composition may includea polypeptide, peptide, or protein that is or is at least 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% identical or similar to an EsxBprotein. In certain aspects the EsxB protein will have all or part ofthe amino acid sequence of SEQ ID NO:12.

In yet still further embodiments of the invention a composition mayinclude a polypeptide, peptide, or protein that is or is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical or similar toan SdrD protein. In certain aspects the SdrD protein will have all orpart of the amino acid sequence of SEQ ID NO:13.

In further embodiments of the invention a composition may include apolypeptide, peptide, or protein that is or is at least 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% identical or similar to an SdrEprotein. In certain aspects the SdrE protein will have all or part ofthe amino acid sequence of SEQ ID NO:14.

In still further embodiments of the invention a composition may includea polypeptide, peptide, or protein that is or is at least 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% identical or similar to an IsdAprotein. In certain aspects the IsdA protein will have all or part ofthe amino acid sequence of SEQ ID NO:15.

In yet still further embodiments of the invention a composition mayinclude a polypeptide, peptide, or protein that is or is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical or similar toan IsdB protein. In certain aspects the IsdB protein will have all orpart of the amino acid sequence of SEQ ID NO:16.

Embodiments of the invention include compositions that include apolypeptide, peptide, or protein that is or is at least 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% identical or similar to a EsaBprotein. In certain aspects the EsaB protein will have all or part ofthe amino acid sequence of SEQ ID NO:17.

In a further embodiments of the invention a composition may include apolypeptide, peptide, or protein that is or is at least 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% identical or similar to a ClfBprotein. In certain aspects the ClfB protein will have all or part ofthe amino acid sequence of SEQ ID NO:18.

In still further embodiments of the invention a composition may includea polypeptide, peptide, or protein that is or is at least 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% identical or similar to an IsdCprotein. In certain aspects the IsdC protein will have all or part ofthe amino acid sequence of SEQ ID NO:19.

In yet further embodiments of the invention a composition may include apolypeptide, peptide, or protein that is or is at least 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% identical or similar to a SasFprotein. In certain aspects the SasF protein will have all or part ofthe amino acid sequence of SEQ ID NO:20.

In yet still further embodiments of the invention a composition mayinclude a polypeptide, peptide, or protein that is or is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical or similar to aSdrC protein. In certain aspects the SdrC protein will have all or partof the amino acid sequence of SEQ ID NO:21.

In yet still further embodiments of the invention a composition mayinclude a polypeptide, peptide, or protein that is or is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical or similar to aClfA protein. In certain aspects the ClfA protein will have all or partof the amino acid sequence of SEQ ID NO:22.

In yet still further embodiments of the invention a composition mayinclude a polypeptide, peptide, or protein that is or is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical or similar toan Eap protein. In certain aspects the Eap protein will have all or partof the amino acid sequence of SEQ ID NO:23.

In yet still further embodiments of the invention a composition mayinclude a polypeptide, peptide, or protein that is or is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical or similar toan Ebh protein. In certain aspects the Ebh protein will have all or partof the amino acid sequence of SEQ ID NO:24.

In yet still further embodiments of the invention a composition mayinclude a polypeptide, peptide, or protein that is or is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical or similar toan Emp protein. In certain aspects the Emp protein will have all or partof the amino acid sequence of SEQ ID NO:25.

In yet still further embodiments of the invention a composition mayinclude a polypeptide, peptide, or protein that is or is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical or similar toan EsaC protein. In certain aspects the EsaC protein will have all orpart of the amino acid sequence of SEQ ID NO:26. Sequence of EsaCpolypeptides can be found in the protein databases and include, but arenot limited to accession numbers ZP_(—)02760162 (GI:168727885),NP_(—)645081.1 (GI:21281993), and NP 370813.1 (GI:15923279), each ofwhich is incorporated herein by reference as of the priority date ofthis application.

In yet still further embodiments of the invention a composition mayinclude a polypeptide, peptide, or protein that is or is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical or similar to aCoa protein. In certain aspects the Coa protein will have all or part ofthe amino acid sequence of SEQ ID NO:27.

In yet still further embodiments of the invention a composition mayinclude a polypeptide, peptide, or protein that is or is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical or similar to aHla protein. In certain aspects the Hla protein will have all or part ofthe amino acid sequence of SEQ ID NO:28.

In yet still further embodiments of the invention a composition mayinclude a polypeptide, peptide, or protein that is or is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical or similar to avWa protein. In certain aspects the vWa protein will have all or part ofthe amino acid sequence of SEQ ID NO:29.

In yet still further embodiments of the invention a composition mayinclude a polypeptide, peptide, or protein that is or is at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical or similar to avWbp protein. In certain aspects the vWbp protein will have all or partof the amino acid sequence of SEQ ID NO:32.

In certain aspects, a polypeptide or segment/fragment can have asequence that is at least 85%, at least 90%, at least 95%, at least 98%,or at least 99% or more identical to the amino acid sequence of thereference polypeptide. The term “similarity” refers to a polypeptidethat has a sequence that has a certain percentage of amino acids thatare either identical with the reference polypeptide or constituteconservative substitutions with the reference polypeptides.

The polypeptides described herein may include 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, or more variant amino acids within at least, orat most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192,193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248,249, 250, 300, 400, 500, 550, 1000 or more contiguous amino acids, orany range derivable therein, of SEQ ID NO:2-30, or SEQ ID NO:32-34.

A polypeptide segment as described herein may include 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196,197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210,211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224,225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238,239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400,500, 550, 1000 or more contiguous amino acids, or any range derivabletherein, of SEQ ID NO:2-30, or SEQ ID NO:33-34.

The compositions may be formulated in a pharmaceutically acceptablecomposition. In certain aspects of the invention the staphylococcusbacterium is an S. aureus bacterium.

In further aspects, a composition may be administered more than one timeto the subject, and may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20 or more times. The administration of the compositions include,but is not limited to oral, parenteral, subcutaneous, intramuscular,intravenous, or various combinations thereof, including inhalation oraspiration.

In still further embodiments, a composition comprises a recombinantnucleic acid molecule encoding a polypeptide described herein orsegments/fragments thereof. Typically a recombinant nucleic acidmolecule encoding a polypeptide described herein contains a heterologouspromoter. In certain aspects, a recombinant nucleic acid molecule of theinvention is a vector, in still other aspects the vector is a plasmid.In certain embodiments the vector is a viral vector. In certain aspectsa composition includes a recombinant, non-staphylococcus bacteriumcontaining or expressing a polypeptide described herein. In particularaspects the recombinant non-staphylococcus bacteria is Salmonella oranother gram-positive bacteria. A composition is typically administeredto mammals, such as human subjects, but administration to other animalsthat are capable of eliciting an immune response is contemplated. Infurther aspects the staphylococcus bacterium containing or expressingthe polypeptide is Staphylococcus aureus. In further embodiments theimmune response is a protective immune response.

In further embodiments a composition comprises a recombinant nucleicacid molecule encoding all or part of one or more of a Eap, Ebh, Emp,EsaB, EsaC, EsxA, EsxB, SdrC, SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, Coa,Hla, IsdC, SasF, SpA, vWbp, or vWh protein or peptide or variantthereof. Additional staphylococcal antigens that can be used incombination with the polypeptides described herein include, but are notlimited to 52 kDa vitronectin binding protein (WO 01/60852), Aaa, Aap,Ant, autolysin glucosaminidase, autolysin amidase, Cna, collagen bindingprotein (U.S. Pat. No. 6,288,214), EFB (FIB), Elastin binding protein(EbpS), EPB, FbpA, fibrinogen binding protein (U.S. Pat. No. 6,008,341),Fibronectin binding protein (U.S. Pat. No. 5,840,846), FnbA, FnbB, GehD(US 2002/0169288), HarA, HBP, Immunodominant ABC transporter, IsaA/PisA, laminin receptor, Lipase GehD, MAP, Mg2+ transporter, MHC IIanalogue (U.S. Pat. No. 5,648,240), MRPII, Npase, RNA III activatingprotein (RAP), SasA, SasB, SasC, SasD, SasK,SBI, SdrF (WO 00/12689),SdrG/Fig (WO 00/12689), SdrH (WO 00/12689), SEA exotoxins (WO 00/02523),SEB exotoxins (WO 00/02523), SitC and Ni ABC transporter,SitC/MntC/saliva binding protein (U.S. Pat. No. 5,801,234), SsaA, SSP-1,SSP-2, and/or Vitronectin binding protein. In particular aspects, abacteria is a recombinant non-staphylococcus bacteria, such as aSalmonella or other gram-positive bacteria.

Compositions of the invention are typically administered to humansubjects, but administration to other animals that are capable ofeliciting an immune response to a staphylococcus bacterium iscontemplated, particularly cattle, horses, goats, sheep and otherdomestic animals, i.e., mammals.

In certain aspects the staphylococcus bacterium is a Staphylococcusaureus. In further embodiments the immune response is a protectiveimmune response. In still further aspects, the methods and compositionsof the invention can be used to prevent, ameliorate, reduce, or treatinfection of tissues or glands, e.g., mammary glands, particularlymastitis and other infections. Other methods include, but are notlimited to prophylactically reducing bacterial burden in a subject notexhibiting signs of infection, particularly those subjects suspected ofor at risk of being colonized by a target bacteria, e.g., patients thatare or will be at risk or susceptible to infection during a hospitalstay, treatment, and/or recovery.

Any embodiment discussed with respect to one aspect of the inventionapplies to other aspects of the invention as well. In particular, anyembodiment discussed in the context of a SpA variant polypeptide orpeptide or nucleic acid may be implemented with respect to otherantigens, such as Eap, Ebh, Emp, EsaC, EsxA, EsxB, SdrC, SdrD, SdrE,IsdA, IsdB, ClfA, ClfB, Coa, Hla, IsdC, SasF, vWbp, vWh, 52 kDavitronectin binding protein (WO 01/60852), Aaa, Aap, Ant, autolysinglucosaminidase, autolysin amidase, Cna, collagen binding protein (U.S.Pat. No. 6,288,214), EFB (FIB), Elastin binding protein (EbpS), EPB,FbpA, fibrinogen binding protein (U.S. Pat. No. 6,008,341), Fibronectinbinding protein (U.S. Pat. No. 5,840,846), FnbA, FnbB, GehD (US2002/0169288), HarA, HBP, Immunodominant ABC transporter, IsaA/P isA,laminin receptor, Lipase GehD, MAP, Mg2+ transporter, MHC II analogue(U.S. Pat. No. 5,648,240), MRPII, Npase, RNA III activating protein(RAP), SasA, SasB, SasC, SasD, SasK,SBI, SdrF (WO 00/12689), SdrG/Fig(WO 00/12689), SdrH (WO 00/12689), SEA exotoxins (WO 00/02523), SEBexotoxins (WO 00/02523), SitC and Ni ABC transporter, SitC/MntC/salivabinding protein (U.S. Pat. No. 5,801,234), SsaA, SSP-1, SSP-2, and/orVitronectin binding protein (or nucleic acids), and vice versa. It isalso understood that any one or more of Eap, Ebh, Emp, EsaC, EsxA, EsxB,SdrC, SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, Coa, Hla, IsdC, SasF, vWbp,vWh, 52 kDa vitronectin binding protein (WO 01/60852), Aaa, Aap, Ant,autolysin glucosaminidase, autolysin amidase, Cna, collagen bindingprotein (US6288214), EFB (FIB), Elastin binding protein (EbpS), EPB,FbpA, fibrinogen binding protein (U.S. Pat. No. 6,008,341), Fibronectinbinding protein (U.S. Pat. No. 5,840,846), FnbA, FnbB, GehD (US2002/0169288), HarA, HBP, Immunodominant ABC transporter, IsaA/P isA,laminin receptor, Lipase GehD, MAP, Mg2+ transporter, MHC II analogue(U.S. Pat. No. 5,648,240), MRPII, Npase, RNA III activating protein(RAP), SasA, SasB, SasC, SasD, SasK,SBI, SdrF (WO 00/12689), SdrG/Fig(WO 00/12689), SdrH (WO 00/12689), SEA exotoxins (WO 00/02523), SEBexotoxins (WO 00/02523), SitC and Ni ABC transporter, SitC/MntC/salivabinding protein (U.S. Pat. No. 5,801,234), SsaA, SSP-1, SSP-2, and/orVitronectin binding protein can be specifically excluded from a claimedcomposition.

Embodiments of the invention include compositions that contain or do notcontain a bacterium. A composition may or may not include an attenuatedor viable or intact staphylococcal bacterium. In certain aspects, thecomposition comprises a bacterium that is not a staphylococcal bacteriumor does not contain staphylococcal bacteria. In certain embodiments abacterial composition comprises an isolated or recombinantly expressedstaphylococcal Protein A variant or a nucleotide encoding the same. Thecomposition may be or include a recombinantly engineered staphylococcusbacterium that has been altered in a way that comprises specificallyaltering the bacterium with respect to a secreted virulence factor orcell surface protein. For example, the bacteria may be recombinantlymodified to express more of the virulence factor or cell surface proteinthan it would express if unmodified.

The term “isolated” can refer to a nucleic acid or polypeptide that issubstantially free of cellular material, bacterial material, viralmaterial, or culture medium (when produced by recombinant DNAtechniques) of their source of origin, or chemical precursors or otherchemicals (when chemically synthesized). Moreover, an isolated compoundrefers to one that can be administered to a subject as an isolatedcompound; in other words, the compound may not simply be considered“isolated” if it is adhered to a column or embedded in an agarose gel.Moreover, an “isolated nucleic acid fragment” or “isolated peptide” is anucleic acid or protein fragment that is not naturally occurring as afragment and/or is not typically in the functional state.

Moieties of the invention, such as polypeptides, peptides, antigens, orimmunogens, may be conjugated or linked covalently or noncovalently toother moieties such as adjuvants, proteins, peptides, supports,fluorescence moieties, or labels. The term “conjugate” or“immunoconjugate” is broadly used to define the operative association ofone moiety with another agent and is not intended to refer solely to anytype of operative association, and is particularly not limited tochemical “conjugation.” Recombinant fusion proteins are particularlycontemplated. Compositions of the invention may further comprise anadjuvant or a pharmaceutically acceptable excipient. An adjuvant may becovalently or non-covalently coupled to a polypeptide or peptide of theinvention. In certain aspects, the adjuvant is chemically conjugated toa protein, polypeptide, or peptide.

The term “providing” is used according to its ordinary meaning toindicate “to supply or furnish for use.” In some embodiments, theprotein is provided directly by administering the protein, while inother embodiments, the protein is effectively provided by administeringa nucleic acid that encodes the protein. In certain aspects theinvention contemplates compositions comprising various combinations ofnucleic acid, antigens, peptides, and/or epitopes.

The subject will have (e.g., are diagnosed with a staphylococcalinfection), will be suspected of having, or will be at risk ofdeveloping a staphylococcal infection. Compositions of the presentinvention include immunogenic compositions wherein the antigen(s) orepitope(s) are contained in an amount effective to achieve the intendedpurpose. More specifically, an effective amount means an amount ofactive ingredients necessary to stimulate or elicit an immune response,or provide resistance to, amelioration of, or mitigation of infection.In more specific aspects, an effective amount prevents, alleviates orameliorates symptoms of disease or infection, or prolongs the survivalof the subject being treated. Determination of the effective amount iswell within the capability of those skilled in the art, especially inlight of the detailed disclosure provided herein. For any preparationused in the methods of the invention, an effective amount or dose can beestimated initially from in vitro studies, cell culture, and/or animalmodel assays. For example, a dose can be formulated in animal models toachieve a desired immune response or circulating antibody concentrationor titer. Such information can be used to more accurately determineuseful doses in humans.

The embodiments in the Example section are understood to be embodimentsof the invention that are applicable to all aspects of the invention.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” It is also contemplatedthat anything listed using the term “or” may also be specificallyexcluded.

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

Following long-standing patent law, the words “a” and “an,” when used inconjunction with the word “comprising” in the claims or specification,denotes one or more, unless specifically noted.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages andobjects of the invention as well as others which will become clear areattained and can be understood in detail, more particular descriptionsand certain embodiments of the invention briefly summarized above areillustrated in the appended drawings. These drawings form a part of thespecification. It is to be noted, however, that the appended drawingsillustrate certain embodiments of the invention and therefore are not tobe considered limiting in their scope.

FIGS. 1A-1E Generation of a non-toxigenic protein A vaccine. FIG. 1ATranslational protein A (SpA) product of S. aureus Newman and USA300 LACwith an N-terminal signal peptide (white box), five immunoglobulinbinding domains (IgBDs designated E, D, A, B and C), variable region Xand C-terminal sorting signal (black box). FIG. 1B, Amino acid sequenceof the five IgBDs as well as nontoxigenic SpA-D_(KKAA), with thepositions of triple α-helical bundles (H1, H2 and H3) as well asglutamine (Q) 9, 10 and aspartate (D) 36, 37 indicated. FIG. 1C,Coomassie Blue-stained SDS-PAGE of SpA, SpA-D, SpA-D_(KKAA) or SrtApurified on Ni-NTA sepharose in the presence or absence of humanimmunoglobulin (hIgG). FIG. 1D, ELISA examining the association ofimmobilized SpA, SpA-D or SpA-D_(KKAA) with human IgG as well as its Fcor F(ab)₂ fragments and von Willebrand factor (vWF). FIG. 1E, CD19+ Blymphocytes in splenic tissue of BALB/c mice that had been mockimmunized or treated with SpA-D or SpA-D_(KKAA) were quantified by FACS.

FIG. 2 Non-toxigenic protein A vaccine prevents abscess formation.Histopathology of renal tissue isolated during necropsy of BALB/c micethat had been mock immunized (PBS) or vaccinated with SpA, SpA-D as wellas SpA-D_(KKAA) and challenged with S. aureus Newman. Thin sectionedtissues were stained with hematoxylin-eosin. White arrows identifypolymorphonuclear leukocyte (PMN) infiltrates. Dark arrows identifystaphylococcal abscess communities.

FIGS. 3A-C Antibodies raised by the non-toxigenic protein A vaccineblock the B cell superantigen function of SpA. FIG. 3A, Rabbitantibodies raised against SpA-D_(KKAA) were purified on a matrix withimmobilized antigen and analyzed by Coomassie Blue-stained SDS-PAGE.Antibodies were cleaved with pepsin and F(ab)₂ fragments were purifiedby a second round of affinity chromatography on SpA-D_(KKAA) matrix.FIG. 3B, SpA-D_(KKAA) specific F(ab)₂ interfere with the binding of SpAor SpA-D to human immunoglobulin (hIgG) or, FIG. 3C, to von WillebrandFactor (vWF).

FIGS. 4A-D Full-length non-toxigenic protein A generates improved immuneresponses. FIG. 4A, Full-length SpA_(KKAA) was purified on Ni-NTAsepharose and analyzed by Coomassie-Blue stained SDS-PAGE. FIG. 4B,CD19+ B lymphocytes in splenic tissue of BALB/c mice that had been mockimmunized or treated with SpA or SpA_(KKAA) were quantified by FACS.FIG. 4C, ELISA examining the association of immobilized SpA orSpA_(KKAA) with human IgG as well as its Fc or F(ab)2 fragments or vonWillebrand factor (vWF). FIG. 4D, Human or mouse serum antibody titersto diphtheria toxoid (CRM197) and non-toxigenic SpA_(KKAA) orSpA-D_(KKAA). Human volunteers with a history of DTaP immunization andstaphylococcal infection (n=16) as well as mice (n=20) that had beeninfected with S. aureus Newman or USA 300 LAC or immunized withSpA_(KKAA) or SpA-D_(KKAA) were examined by quantitative dot blot.

FIG. 5 Protein A is required for the pathogenesis of lethal S. aureusinfections in mice. Cohorts of BALB/c mice (n=8) were injected withsuspensions of 2×10⁸ CFU S. aureus Newman or its isogenic protein Adeletion variant (Δspa) in PBS. Infected animals were monitored forsurvival over a period of 15 days.

FIGS. 6A-B Antibodies against protein A protect mice against lethal S.aureus infections. FIG. 6A Cohorts of BALB/c mice (n=10) were injectedwith 5 mg kg⁻¹ affinity purified rabbit IgG specific for SpA_(KKAA)(α-Sp_(AKKAA)) or the plague vaccine antigen rV10 (DeBord et al., 2006)(mock). Four hours later, each animal was infected by intraperitonealinjection with a suspension of 3×10⁸ CFU S. aureus Newman and monitoredfor survival over a period of 10 days. Data are representative of threeindependent experiments FIG. 6B Cohorts of BALB/c mice (n=10) wereprime-booster immunized with SpA_(KKAA) or PBS/adjuvant control (mock).Each animal was subsequently infected by intraperitoneal injection witha suspension of 6×10⁸ CFU S. aureus Newman and monitored for survivalover a period of 10 days. Statistical significance (P) was analyzed withthe unpaired two-tailed log-rank test. Data are representative of allthree independent experiments.

FIG. 7 SpA_(KKAA) immunization protects mice against challenge with thevancomycin-resistant MRSA isolated Mu50. Cohorts of BALB/c mice (n=15)were prime-booster immunized with SpA_(KKAA) or PBS/adjuvant control(mock). Each animal was subsequently infected by intravenous injectionwith a suspension of 3×10⁷ CFU S. aureus Mu50. Staphylococcal load,calculated as log 10 CFU g⁻¹, was determined in homogenized renaltissues 4 days following infection. Statistical significance wascalculated with the unpaired two-tailed Students t-test and P-valuerecorded.

FIGS. 8A-B Lack of protective immune responses to staphylococcalinfections. FIG. 8A Staphylococcal infection does not generateprotective immunity. BALB/c mice (n=10) were infected with S. aureusNewman or mock challenged (PBS) for thirty days and infection clearedwith chloramphenicol treatment. Both cohorts of animals were thenchallenged with S. aureus Newman and bacterial load (CFU) in kidneytissue homogenate analyzed following necropsy on day 4. Data arerepresentative of three independent analyses. FIG. 8B IsdB immunizationdoes not protect mice against S. aureus USA300 (LAC) challenge. BALB/cmice (n=10) were immunized with IsdB (100 μg IsdB emulsified in CFAfollowed by IFA/IsdB booster on day 11) and challenged by retro-orbitalinjection with 5×10⁶ CFU S. aureus USA300 (LAC) on day 21. Four daysfollowing challenge, kidneys were removed during necropsy andstaphylococcal load per gram of homogenized tissue enumerated by colonyformation on agar plates. Compared to mock immunized (PBS/adjuvant)animals with 6.93 (±0.24) log 10 CFU g⁻¹, IsdB vaccination wasassociated with 6.25 (±0.46) log 10 CFU g⁻¹ and did not generatestatistically significant protection (P=0.2138, two-tailed Student'st-test) from USA300 (LAC) challenge. Data are representative of threeindependent analyses.

FIG. 9 Comparison of abscess formation in mice treated with PBS, SpA,SpA-D and SpA-D_(KKAA).

FIGS. 10A-10H Localization of prothrombin, fibrinogen, coagulase (Coa),and von Willebrand factor binding protein (vWbp) in staphylococcalabscesses. BALB/c mice infected by intravenous inoculation with 1×10⁷CFU S. aureus Newman were killed 5 days post infection. Kidneys wereremoved, embedded in paraffin, thin-sectioned and stained byimmunochemistry using rabbit antibodies (a) specific for mouseprothrombin (FIGS. 10A, 10C), mouse fibrinogen/fibrin (FIGS. 10B, 10D),S. aureus Coa (FIGS. 10E, 10G) or S. aureus vWbp (FIGS. 10F, 10H).Displayed images are representative of three sampled kidneys. PanelsFIGS. 10C, 10D, 10G, and 10H illustrate antibody staining within asingle abscess analyzed as four sequential sections, enlarged from anarea in panels FIGS. 10A, 10B, 10E, and 10F that is defined by box withwhite margins.

FIGS. 11A-11C Staphylococcus aureus coa and vWbp mutants display defectsin blood clotting. (FIG. 11A) Diagram illustrating the primarytranslational product of coa and vWbp including signal peptide (S), theD1 and D2 domain from prothrombin binding, a domain of unknown function,von Willebrand factor (vWF) binding site on vWbp, and the fibrinogenbinding repeats (R) of Coa. Numbers indicate amino acid residues. (FIG.11B) Culture supernatants from S. aureus Newman (wild-type) or isogenicvariants lacking coa (Δcoa), vWbp (ΔvWbp) or both genes (Δcoa, ΔvWbp)were examined by immunoblotting with antibodies specific for Coa (αCoa)or vWbp (αvWbp). For complementation studies, plasmids expressing thewild-type alleles of coa (pcoa) or vWbp (pvWbp) were electroporated intostaphylococcal strains and subsequently analyzed by immunoblotting.(FIG. 11C) Lepirudin-treated mouse blood was mock treated or infectedwith S. aureus Newman or its isogenic coagulase variants and incubatedfor up to 48 hours at 25° C. Tubes were tilted to assess forcoagulation. Data are representative of four independent determinations.

FIGS. 12A-12R Contributions of coa and vWbp to bacterial survival inblood and S. aureus induced lethal bacteremia of mice. (FIG. 12A)Staphylococcal strains Newman, Δcoa, ΔvWbp or Δcoa, ΔvWbp and thecomplemented variants were incubated with lepirudin anticoagulated mouseblood for 30 minutes and bacterial survival assessed by colony formationon agar plates. Data were generated from three separate trials. (FIG.12B) Cohorts of 10 mice were injected into the retro-orbital plexus with1×10⁸ CFU of S. aureus Newman (wild-type) as well as Δcoa, ΔvWbp orΔcoa, ΔvWbp. Animal survival over time was recorded over 10 days.Similar to B, mice were given 1×10⁷ CFU of staphylococcal strains Newman(FIGS. 12C, E and K, M), ΔvWbp (FIGS. 14D, F and M, L), Δcoa (FIGS. 14G,I and O, Q) or Δcoa, ΔvWbp (FIGS. 12H, J and P, R), harvested on days 5(FIG. 12C-J) or 15 (FIG. 12K-R) and assessed for bacterial load in therenal tissue (Table 7) and histopathological abscess formation. Allanimal data are representative of two independent experiments.

FIGS. 13A-13D Antibodies against Coa and vWbp block the clotting ofblood by staphylococcal coagulases. (FIG. 13A) His₆-Coa and His₆-vWbpwere purified by affinity chromatography from E. coli and analyzed onCoomassie-stained SDS-PAGE. (FIG. 13B) Rabbit antibodies raised againstHis₆-Coa or His₆-vWbp were affinity purified and analyzed by ELISA forimmune reactivity with purified coagulases. Data are averaged from threeindependent experimental determinations. (FIG. 13C) Lepirudin-treatedmouse blood was treated with PBS (mock), irrelevant antibodies (αV10) orantibodies directed against Coa (αCoa), vWbp (αvWbp) or both coagulases(αCoa/αvWbp) prior to infection with S. aureus Newman and incubation for48 hours at 25° C. (FIG. 13D) Lepirudin-treated mouse blood was treatedwith antibodies as above. Blood samples were then incubated withfunctionally active Coa or vWbp and coagulation time recorded.

FIGS. 14A-14F Biological effects of antibodies directed againststaphylococcal coagulases. Surface plasmon resonance measurement ofantibody perturbing the association between Coa or vWbp and prothrombinor fibrinogen. Response differences upon addition of coagulase (Coa) toeither prothrombin (FIG. 14A) or fibrinogen (FIG. 14B) were compared toresponse differences in the presence of increasing amounts of antibodies(αCoa—1:1, 1:2, 1:4, 1:8). Response differences upon addition of vWbp toeither prothrombin (FIG. 16A) or fibrinogen (FIG. 14B) were compared toresponse differences in the presence of increasing amounts of antibodies(αvWbp—1:1, 1:2, 1:4, 1:8). (FIGS. 14E, F) Purified active Coa or vWbpwas incubated in a 1:1 molar ratio with human prothrombin. The enzymaticability of the complex was assessed by monitoring the rate of S-2238cleavage (fibrinogen substitute chromogenic substrate, given in excess).The assay was repeated in presence of specific or cross antibodies addedin 3M excess and the data was normalized to the % average activitywithout inhibition. Data are an average of three independent trials.

FIG. 15 Contribution of coagulase specific antibodies to the survival ofmice with staphylococcal bacteremia. Twenty-four hours prior toinfection, BALB/c mice (n=15) were injected into the peritoneum withpurified rabbit antibodies (5 mg antibody/kg body weight). Animals werethen challenged with 1×10⁸ CFU S. aureus Newman injected into theretro-orbital plexus and monitored for survival. Data are representativeof two independent experiments.

FIGS. 16A-16H Passive transfer of coagulase antibodies confersprotection against S. aureus abscess formation. An experimental mock(PBS, FIGS. 18A and 18C) or purified rabbit antibodies directed againstvWbp (αvWbp, FIGS. 18B and 18D), Coa (αCoa, FIGS. 18E and 18G) or bothcoagulases (αCoa/αvWbp, FIGS. 18F and 18H) were injected into theperitoneal cavity of BALB/c mice (n=10) and antibody titers analyzed byELISA (Table 8). Passively immunized animals were infected by injecting1×10⁷CFU S. aureus Newman into the retro-orbital plexus. Bacterial loadand abscess formation were determined following necropsy in the kidneysof animals that had been killed five days following infection. Renaltissues were fixed with paraformaldehyde, embedded in paraffin, thinsectioned, stained with hematoxylin-eosin and histopathology imagesacquired by light microscopy. Data are representative of two separateexperiments.

FIG. 17 s A-H Immunization with coagulases protects mice against S.aureus abscess formation. BALB/c mice (n=15) were immunized with 50 μgHis₆-Coa, His₆-vWbp, His₆-Coa and His₆-vWbp or mock (PBS) emulsifiedwith adjuvant on day 0 and 11 and antibody titers analyzed by ELISA onday 21 (Table 8). On day 21, animals were challenged by injecting1×10⁷CFU S. aureus Newman into the retro-orbital plexus. Bacterial loadand abscess formation were determined following necropsy in the kidneysof animals that had been killed five days following infection. Renaltissues were fixed with paraformaldehyde, embedded in paraffin, thinsectioned, stained with hematoxylin-eosin and histopathology imagesacquired by light microscopy. Data are representative of two separateexperiments.

DETAILED DESCRIPTION

Staphylococcus aureus is a commensal of the human skin and nares, andthe leading cause of bloodstream, skin and soft tissue infections(Klevens et al., 2007). Recent dramatic increases in the mortality ofstaphylococcal diseases are attributed to the spread ofmethicillin-resistant S. aureus (MRSA) strains often not susceptible toantibiotics (Kennedy et al., 2008). In a large retrospective study, theincidence of MRSA infections was 4.6% of all hospital admissions in theUnited States (Klevens et al., 2007). The annual health care costs for94,300 MRSA infected individuals in the United States exceed $2.4billion (Klevens et al., 2007). The current MRSA epidemic hasprecipitated a public health crisis that needs to be addressed bydevelopment of a preventive vaccine (Boucher and Corey, 2008). To date,an FDA licensed vaccine that prevents S. aureus diseases is notavailable.

The inventors describe here the use of Protein A, a cell wall anchoredsurface protein of staphylococci, for the generation of variants thatcan serve as subunit vaccines. The pathogenesis of staphylococcalinfections is initiated as bacteria invade the skin or blood stream viatrauma, surgical wounds, or medical devices (Lowy, 1998). Although theinvading pathogen may be phagocytosed and killed, staphylococci can alsoescape innate immune defenses and seed infections in organ tissues,inducing inflammatory responses that attract macrophages, neutrophils,and other phagocytes (Lowy, 1998). The responsive invasion of immunecells to the site of infection is accompanied by liquefaction necrosisas the host seeks to prevent staphylococcal spread and allow for removalof necrotic tissue debris (Lam et al., 1963). Such lesions can beobserved by microscopy as hypercellular areas containing necrotictissue, leukocytes, and a central nidus of bacteria (Lam et al., 1963).Unless staphylococcal abscesses are surgically drained and treated withantibiotics, disseminated infection and septicemia produce a lethaloutcome (Sheagren, 1984).

Staphylococcal Antigens

A. Staphylcoccal Protein A (SpA)

All Staphylococcus aureus strains express the structural gene forProtein A (spa) (Jensen, 1958; Said-Salim et al., 2003), a wellcharacterized virulence factor whose cell wall anchored surface proteinproduct (SpA) encompasses five highly homologous immunoglobulin bindingdomains designated E, D, A, B, and C (Sjodahl, 1977). These domainsdisplay ˜80% identity at the amino acid level, are 56 to 61 residues inlength, and are organized as tandem repeats (Uhlen et al., 1984). SpA issynthesized as a precursor protein with an N-terminal YSIRK/GS signalpeptide and a C-terminal LPXTG motif sorting signal (DeDent et al.,2008; Schneewind et al., 1992). Cell wall anchored Protein A isdisplayed in great abundance on the staphylococcal surface (DeDent etal., 2007; Sjoquist et al., 1972). Each of its immunoglobulin bindingdomains is composed of anti-parallel α-helices that assemble into athree helix bundle and bind the Fc domain of immunoglobulin G (IgG)(Deisenhofer, 1981; Deisenhofer et al., 1978), the VH3 heavy chain (Fab)of IgM (i.e., the B cell receptor) (Graille et al., 2000), the vonWillebrand factor at its A1 domain [vWF A1 is a ligand for platelets](O'Seaghdha et al., 2006) and the tumor necrosis factor α (TNF-α)receptor I (TNFRI) (Gomez et al., 2006), which is displayed on surfacesof airway epithelia (Gomez et al., 2004; Gomez et al., 2007).

SpA impedes neutrophil phagocytosis of staphylococci through itsattribute of binding the Fc component of IgG (Jensen, 1958; Uhlen etal., 1984). Moreover, SpA is able to activate intravascular clotting viaits binding to von Willebrand factor AI domains (Hartleib et al., 2000).Plasma proteins such as fibrinogen and fibronectin act as bridgesbetween staphylococci (CIfA and CIfB) and the platelet integrinGPIIb/IIIa (O'Brien et al., 2002), an activity that is supplementedthrough Protein A association with vWF AI, which allows staphylococci tocapture platelets via the GPIb-α platelet receptor (Foster, 2005;O'Seaghdha et al., 2006). SpA also binds TNFRI and this interactioncontributes to the pathogenesis of staphylococcal pneumonia (Gomez etal., 2004). SpA activates proinflammatory signaling through TNFR1mediated activation of TRAF2, the p38/c-Jun kinase, mitogen activateprotein kinase (MAPK) and the Rel-transcription factor NF-KB. SpAbinding further induces TNFR1 shedding, an activity that appears torequire the TNF-converting enzyme (TACE)(Gomez et al., 2007). All of theaforementioned SpA activities are mediated through its five IgG bindingdomains and can be perturbed by the same amino acid substitutions,initially defined by their requirement for the interaction betweenProtein A and human IgG1 (Cedergren et al., 1993.

SpA also functions as a B cell superantigen by capturing the Fab regionof VH3 bearing IgM, the B cell receptor (Gomez et al., 2007; Goodyear etal., 2003; Goodyear and Silverman, 2004; Roben et al., 1995). Followingintravenous challenge, staphylococcal Protein A (SpA) mutations show areduction in staphylococcal load in organ tissues and dramaticallydiminished ability to form abscesses (described herein). Duringinfection with wildtype S. aureus, abscesses are formed withinforty-eight hours and are detectable by light microscopy ofhematoxylin-eosin stained, thin-sectioned kidney tissue, initiallymarked by an influx of polymorphonuclear leukocytes (PMNs). On day 5 ofinfection, abscesses increase in size and enclosed a central populationof staphylococci, surrounded by a layer of eosinophilic, amorphousmaterial and a large cuff of PMNs. Histopathology revealed massivenecrosis of PMNs in proximity to the staphylococcal nidus at the centerof abscess lesions as well as a mantle of healthy phagocytes. Theinventors also observed a rim of necrotic PMNs at the periphery ofabscess lesions, bordering the eosinophilic pseudocapsule that separatedhealthy renal tissue from the infectious lesion. Staphylococcal variantslacking Protein A are unable to establish the histopathology features ofabscesses and are cleared during infection.

In previous studies, Cedergren et al. (1993) engineered five individualsubstitutions in the Fc fragment binding sub-domain of the B domain ofSpA, L17D, N28A, I31A and K35A. These authors created these proteins totest data gathered from a three dimensional structure of a complexbetween one domain of SpA and Fc₁. Cedergren et al. determined theeffects of these mutations on stability and binding, but did notcontemplate use of such substitutions for the production of a vaccineantigen.

Brown et al. (1998) describe studies designed to engineer new proteinsbased on SpA that allow the use of more favorable elution conditionswhen used as affinity ligands. The mutations studied included singlemutations of Q13A, Q14H, N15A, N15H, F17H, Y18F, L21H, N32H, or K39H.Brown et al. report that Q13A, N15A, N15H, and N32H substitutions madelittle difference to the dissociation constant values and that the Y18Fsubstitution resulted in a 2 fold decrease in binding affinity ascompared to wild type SpA. Brown et al. also report that L21H and F17Hsubstitutions decrease the binding affinity by five-fold and ahundred-fold respectively. The authors also studied analogoussubstitutions in two tandem domains. Thus, the Brown et al. studies weredirected to generating a SpA with a more favorable elution profile,hence the use of H is substitutions to provide a pH sensitive alterationin the binding affinity. Brown et al. is silent on the use of SpA as avaccine antigen.

Graille et al. (2000) describe a crystal structure of domain D of SpAand the Fab fragment of a human IgM antibody. Graille et al. define byanalysis of a crystal structure the D domain amino acid residues thatinteract with the Fab fragment as residues Q26, G29, F30, Q32, S33, D36,D37, Q40, N43, E47, or L51, as well as the amino acid residues that formthe interface between the domain D sub-domains. Graille et al. definethe molecular interactions of these two proteins, but is silent inregard to any use of substitutions in the interacting residues inproducing a vaccine antigen.

O'Seaghdha et al. (2006) describe studies directed at elucidating whichsub-domain of domain D binds vWF. The authors generated single mutationsin either the Fc or VH3 binding sub-domains, i.e., amino acid residuesF5A, Q9A, Q10A, F13A, Y14A, L17A, N28A, I31A, K35A, G29A, F30A, S33A,D36A, D37A, Q40A, E47A, or Q32A. The authors discovered that vWF bindsthe same sub-domain that binds Fc. O'Seaghda et al. define thesub-domain of domain D responsible for binding vWF, but is silent inregard to any use of substitutions in the interacting residues inproducing a vaccine antigen.

Gomez et al. (2006) describe the identification of residues responsiblefor activation of the TNFR1 by using single mutations of F5A, F13A,Y14A, L17A, N21A, I31A, Q32A, and K35A. Gomez et al. is silent in regardto any use of substitutions in the interacting residues in producing avaccine antigen.

Recombinant affinity tagged Protein A, a polypeptide encompassing thefive IgG domains (EDCAB) (Sjodahl, 1977) but lacking the C-terminalRegion X (Guss et al., 1984), was purified from recombinant E. coli andused as a vaccine antigen (Stranger-Jones et al., 2006). Because of theattributes of SpA in binding the Fc portion of IgG, a specific humoralimmune response to Protein A could not be measured (Stranger-Jones etal., 2006). The inventors have overcome this obstacle through thegeneration of SpA-DQ9,10K;D36,37A. BALB/c mice immunized withrecombinant Protein A (SpA) displayed significant protection againstintravenous challenge with S. aureus strains: a 2.951 log reduction instaphylococcal load as compared to the wild-type (P>0.005; Student'st-test) (Stranger-Jones et al., 2006). SpA specific antibodies may causephagocytic clearance prior to abscess formation and/or impact theformation of the aforementioned eosinophilic barrier in abscesses thatseparate staphylococcal communities from immune cells since these do notform during infection with Protein A mutant strains. Each of the fiveSpA domains (i.e., domains formed from three helix bundles designated E,D, A, B, and C) exerts similar binding properties (Jansson et al.,1998). The solution and crystal structure of the domain D has beensolved both with and without the Fc and VH3 (Fab) ligands, which bindProtein A in a non-competitive manner at distinct sites (Graille et al.,2000). Mutations in residues known to be involved in IgG binding (FS,Q9, Q10, S11, F13, Y14, L17, N28, I31 and K35) are also required for vWFA1 and TNFR1 binding (Cedergren et al., 1993; Gomez et al., 2006;O'Seaghdha et al., 2006), whereas residues important for the VH3interaction (Q26, G29, F30, S33, D36, D37, Q40, N43, E47) appear to haveno impact on the other binding activities (Graille et al., 2000; Janssonet al., 1998). SpA specifically targets a subset of B cells that expressVH3 family related IgM on their surface, i.e., VH3 type B cell receptors(Roben et al., 1995). Upon interaction with SpA, these B cellsproliferate and commit to apoptosis, leading to preferential andprolonged deletion of innate-like B lymphocytes (i.e., marginal zone Bcells and follicular B2 cells) (Goodyear et al., 2003; Goodyear et al.,2004).

Molecular basis of Protein A surface display and function. Protein A issynthesized as a precursor in the bacterial cytoplasm and secreted viaits YSIRK signal peptide at the cross wall, i.e. the cell divisionseptum of staphylococci (FIG. 1) (DeDent et al., 2007; DeDent et al.,2008). Following cleavage of the C-terminal LPXTG sorting signal,Protein A is anchored to bacterial peptidoglycan crossbridges by sortaseA (Mazmanian et al., 1999; Schneewind et al., 1995; Mazmanian et al.,2000). Protein A is the most abundant surface protein of staphylococci;the molecule is expressed by virtually all S. aureus strains (Cespedeset al., 2005; Kennedy et al., 2008; Said-Salim et al., 2003).Staphylococci turn over 15-20% of their cell wall per division cycle(Navarre and Schneewind, 1999). Murine hydrolases cleave the glycanstrands and wall peptides of peptidoglycan, thereby releasing Protein Awith its attached C-terminal cell wall disaccharide tetrapeptide intothe extracellular medium (Ton-That et al., 1999). Thus, by physiologicaldesign, Protein A is both anchored to the cell wall and displayed on thebacterial surface but also released into surrounding tissues during hostinfection (Marraffini et al., 2006).

Protein A captures immunoglobulins on the bacterial surface and thisbiochemical activity enables staphylococcal escape from host innate andacquired immune responses (Jensen, 1958; Goodyear et al., 2004).Interestingly, region X of Protein A (Guss et al., 1984), a repeatdomain that tethers the IgG binding domains to the LPXTG sortingsignal/cell wall anchor, is perhaps the most variable portion of thestaphylococcal genome (Said-Salim, 2003; Schneewind et al., 1992). Eachof the five immunoglobulin binding domains of Protein A (SpA), formedfrom three helix bundles and designated E, D, A, B, and C, exertssimilar structural and functional properties (Sjodahl, 1977; Jansson etal., 1998). The solution and crystal structure of the domain D has beensolved both with and without the Fc and V_(H)3 (Fab) ligands, which bindProtein A in a non-competitive manner at distinct sites (Graille 2000).

In the crystal structure complex, the Fab interacts with helix II andhelix III of domain D via a surface composed of four VH region β-strands(Graille 2000). The major axis of helix II of domain D is approximately50° to the orientation of the strands, and the interhelical portion ofdomain D is most proximal to the C0 strand. The site of interaction onFab is remote from the Ig light chain and the heavy chain constantregion. The interaction involves the following domain D residues: Asp-36of helix II, Asp-37 and Gln-40 in the loop between helix II and helixIII and several other residues (Graille 2000). Both interacting surfacesare composed predominantly of polar side chains, with three negativelycharged residues on domain D and two positively charged residues on the2A2 Fab buried by the interaction, providing an overall electrostaticattraction between the two molecules. Of the five polar interactionsidentified between Fab and domain D, three are between side chains. Asalt bridge is formed between Arg-H19 and Asp-36 and two hydrogen bondsare made between Tyr-H59 and Asp-37 and between Asn-H82a and Ser-33.Because of the conservation of Asp-36 and Asp-37 in all five IgG bindingdomains of Protein A, the inventors mutated these residues.

The SpA-D sites responsible for Fab binding are structurally separatefrom the domain surface that mediates Fcγ binding. The interaction ofFcγ with domain D primarily involves residues in helix I with lesserinvolvement of helix II (Gouda et al., 1992; Deisenhofer, 1981). Withthe exception of the Gln-32, a minor contact in both complexes, none ofthe residues that mediate the Fcγ interaction are involved in Fabbinding. To examine the spatial relationship between these differentIg-binding sites, the SpA domains in these complexes have beensuperimposed to construct a model of a complex between Fab, theSpA-domain D, and the Fcγ molecule. In this ternary model, Fab and Fcγform a sandwich about opposite faces of the helix II without evidence ofsteric hindrance of either interaction. These findings illustrate how,despite its small size (i.e., 56-61 aa), an SpA domain cansimultaneously display both activities, explaining experimental evidencethat the interactions of Fab with an individual domain arenoncompetitive. Residues for the interaction between SpA-D and Fcγ areGln-9 and Gln-10.

In contrast, occupancy of the Fc portion of IgG on the domain D blocksits interaction with vWF A1 and probably also TNFR1 (O'Seaghdha et al.,2006). Mutations in residues essential for IgG Fc binding (F5, Q9, Q10,S11, F13, Y14, L17, N28, I31 and K35) are also required for vWF A1 andTNFR1 binding (O'Seaghdha et al., 2006; Cedergren et al., 1993; Gomez etal., 2006), whereas residues critical for the VH3 interaction (Q26, G29,F30, S33, D36, D37, Q40, N43, E47) have no impact on the bindingactivities of IgG Fc, vWF A1 or TNFR1 (Jansson et al., 1998; Graille etal., 2000). The Protein A immunoglobulin Fab binding activity targets asubset of B cells that express V_(H)3 family related IgM on theirsurface, i.e., these molecules function as VH3type B cell receptors(Roben et al., 1995). Upon interaction with SpA, these B cells rapidlyproliferate and then commit to apoptosis, leading to preferential andprolonged deletion of innate-like B lymphocytes (i.e., marginal zone Bcells and follicular B2 cells) (Goodyear and Silverman, 2004; Goodyearand Silverman, 2003). More than 40% of circulating B cells are targetedby the Protein A interaction and the V_(H)3 family represents thelargest family of human B cell receptors to impart protective humoralresponses against pathogens (Goodyear and Silverman, 2004; Goodyear andSilverman, 2003). Thus, Protein A functions analogously tostaphylococcal superantigens (Roben et al., 1995), albeit that thelatter class of molecules, for example SEB, TSST-1, TSST-2, formcomplexes with the T cell receptor to inappropriately stimulate hostimmune responses and thereby precipitating characteristic diseasefeatures of staphylococcal infections (Roben et al., 1995; Tiedemann etal., 1995). Together these findings document the contributions ofProtein A in establishing staphylococcal infections and in modulatinghost immune responses.

In sum, Protein A domains can viewed as displaying two differentinterfaces for binding with host molecules and any development ofProtein A based vaccines must consider the generation of variants thatdo not perturb host cell signaling, platelet aggregation, sequestrationof immunoglobulins or the induction of B cell proliferation andapoptosis. Such Protein A variants should also be useful in analyzingvaccines for the ability of raising antibodies that block theaforementioned SpA activities and occupy the five repeat domains attheir dual binding interfaces. This goal is articulated and pursued herefor the first time and methods are described in detail for thegeneration of Protein A variants that can be used as a safe vaccine forhumans. To perturb IgG Fcγ, vWF A1 and TNFR1 binding, glutamine (Q) 9and 10 [numbering derived from the SpA domain D as described in Uhlen etal., 1984] were mutated, and generated lysine substitutions for bothglutamines with the expectation that these abolish the ligand attributesat the first binding interface. To perturb IgM Fab VH3 binding,aspartate (D) 36 and 37 were mutated, each of which is required for theassociation with the B cell receptor. D36 and D37 were both substitutedwith alanine Q9,10K and D36,37A mutations are here combined in therecombinant molecule SpA-DQ9,10K;D36,37A and tested for the bindingattributes of Protein A. Further, SpA-D and SpA-DQ9,10K;D36,37A aresubjected to immunization studies in mice and rabbits and analyzed for[1] the production of specific antibodies (SpA-D Ab); [2] the ability ofSpA-D Ab to block the association between Protein A and its fourdifferent ligands; and, [3] the attributes of SpA-D Ab to generateprotective immunity against staphylococcal infections. (See Examplessection below).

B. Staphylococcal Coagulases

Coagulases are enzymes produced by Staphylococcus bacteria that convertfibrinogen to fibrin. Coa and vW_(h) activate prothrombin withoutproteolysis (Friedrich et al., 2003). The coagulase•prothrombin complexrecognizes fibrinogen as a specific substrate, converting it directlyinto fibrin. The crystal structure of the active complex revealedbinding of the D1 and D2 domains to prothrombin and insertion of itsIle1-Val² N-terminus into the Ile¹⁶ pocket, inducing a functional activesite in the zymogen through conformational change (Friedrich et al.,2003). Exosite I of α-thrombin, the fibrinogen recognition site, andproexosite I on prothrombin are blocked by the D2 of Coa (Friedrich etal., 2003). Nevertheless, association of the tetrameric(Coa•prothrombin)₂ complex binds fibrinogen at a new site with highaffinity (Panizzi et al., 2006). This model explains the coagulantproperties and efficient fibrinogen conversion by coagulase (Panizzi etal., 2006).

Fibrinogen is a large glycoprotein (Mr ˜340,000), formed by three pairsof Aα-, Bβ-, and γ-chains covalently linked to form a “dimer oftrimers,” where A and B designate the fibrinopeptides released bythrombin cleavage (Panizzi et al., 2006). The elongated molecule foldsinto three separate domains, a central fragment E that contains theN-termini of all six chains and two flanking fragments D formed mainlyby the C-termini of the Bβ- and γ-chains. These globular domains areconnected by long triple-helical structures. Coagulase-prothrombincomplexes, which convert human fibrinogen to the self-polymerizingfibrin, are not targeted by circulating thrombin inhibitors (Panizzi etal., 2006). Thus, staphylococcal coagulases bypass the physiologicalblood coagulation pathway.

All S. aureus strains secrete coagulase and vWbp (Bjerketorp et al.,2004; Field and Smith, 1945). Although early work reported importantcontributions of coagulase to the pathogenesis of staphylococcalinfections (Ekstedt and Yotis, 1960; Smith et al., 1947), more recentinvestigations with molecular genetics tools challenged this view byobserving no virulence phenotypes with endocarditis, skin abscess andmastitis models in mice (Moreillon et al., 1995; Phonimdaeng et al.,1990). Generating isogenic variants of S. aureus Newman, a fullyvirulent clinical isolate (Duthie et al., 1952), it is described hereinthat coa mutants indeed display virulence defects in a lethal bacteremiaand renal abscess model in mice. In the inventors experience, S. aureus8325-4 is not fully virulent and it is presumed that mutational lesionsin this strain may not be able to reveal virulence defects in vivo.Moreover, antibodies raised against Coa or vWbp perturb the pathogenesisof S. aureus Newman infections to a degree mirroring the impact of genedeletions. Coa and vWbp contribute to staphylococcal abscess formationand lethal bacteremia and may also function as protective antigens insubunit vaccines.

Biochemical studies document the biological value of antibodies againstCoa and vWbp. By binding to antigen and blocking its association withclotting factors, the antibodies prevent the formation of Coaprothrombinand vWbp•prothrombin complexes. Passive transfer studies revealedprotection of experimental animals against staphylococcal abscessformation and lethal challenge by Coa and vWbp antibodies. Thus, Coa andvWbp neutralizing antibodies generate immune protection againststaphylococcal disease.

Earlier studies revealed a requirement of coagulase for resistingphagocytosis in blood (Smith et al., 1947) and the inventors observed asimilar phenotype for Δcoa mutants in lepirudin-treated mouse blood (seeExample 3 below). As vWbp displays higher affinity for human prothrombinthan the mouse counterpart, it is suspected the same may be true forΔvWbp variants in human blood. Further, expression of Coa and vWbp inabscess lesions as well as their striking distribution in theeosinophilic pseudocapsule surrounding (staphylococcal abscesscommunities (SACs) or the peripheral fibrin wall, suggest that secretedcoagulases contribute to the establishment of these lesions. Thishypothesis was tested and, indeed, Δcoa mutants were defective in theestablishment of abscesses. A corresponding test, blocking Coa functionwith specific antibodies, produced the same effect. Consequently, it isproposed that the clotting of fibrin is a critical event in theestablishment of staphylococcal abscesses that can be targeted for thedevelopment of protective vaccines. Due to their overlapping function onhuman prothrombin, both Coa and vWbp are considered excellent candidatesfor vaccine development.

C. Other Staphylococcal Antigens

Research over the past several decades identified S. aureus exotoxins,surface proteins and regulatory molecules as important virulence factors(Foster, 2005; Mazmanian et al., 2001; Novick, 2003). Much progress hasbeen achieved regarding the regulation of these genes. For example,staphylococci perform a bacterial census via the secretion ofauto-inducing peptides that bind to a cognate receptor at thresholdconcentration, thereby activating phospho-relay reactions andtranscriptional activation of many of the exotoxin genes (Novick, 2003).The pathogenesis of staphylococcal infections relies on these virulencefactors (secreted exotoxins, exopolysaccharides, and surface adhesins).The development of staphylococcal vaccines is hindered by themultifaceted nature of staphylococcal invasion mechanisms. It is wellestablished that live attenuated micro-organisms are highly effectivevaccines; immune responses elicited by such vaccines are often ofgreater magnitude and of longer duration than those produced bynon-replicating immunogens. One explanation for this may be that liveattenuated strains establish limited infections in the host and mimicthe early stages of natural infection. Embodiments of the invention aredirected to compositions and methods including variant SpA polypeptidesand peptides, as well as other immunogenic extracellular proteins,polypeptides, and peptides (including both secreted and cell surfaceproteins or peptides) of gram positive bacteria for the use inmitigating or immunizing against infection. In particular embodimentsthe bacteria is a staphylococcus bacteria. Extracellular proteins,polypeptides, or peptides include, but are not limited to secreted andcell surface proteins of the targeted bacteria.

The human pathogen S. aureus secretes EsxA and EsxB, two ESAT-6 likeproteins, across the bacterial envelope (Burts et al., 2005, which isincorporated herein by reference). Staphylococcal esxA and esxB areclustered with six other genes in the order of transcription: esxA esaAessA esaB essB essC esaC esxB. The acronyms esa, ess, and esx stand forESAT-6 secretion accessory, system, and extracellular, respectively,depending whether the encoded proteins play an accessory (esa) or direct(ess) role for secretion, or are secreted (esx) in the extracellularmilieu. The entire cluster of eight genes is herein referred to as theEss cluster. EsxA, esxB, essA, essB, and essC are all required forsynthesis or secretion of EsxA and EsxB. Mutants that fail to produceEsxA, EsxB, and EssC display defects in the pathogenesis of S. aureusmurine abscesses, suggesting that this specialized secretion system maybe a general strategy of human bacterial pathogenesis. Secretion ofnon-WXG100 substrates by the ESX-1 pathway has been reported for severalantigens including EspA, EspB, Rv3483c, and Rv3615c (Fortune et al.,2005; MacGurn et al., 2005; McLaughlin et al., 2007; Xu et al., 2007).The alternate ESX-5 pathway has also been shown to secrete both WXG100and non-WXG100 proteins in pathogenic mycobacteria (Abdallah et al.,2007; Abdallah et al., 2006).

The Staphylococcus aureus Ess pathway can be viewed as a secretionmodule equipped with specialized transport components (Ess), accessoryfactors (Esa) and cognate secretion substrates (Esx). EssA, EssB andEssC are required for EsxA and EsxB secretion. Because EssA, EssB andEssC are predicted to be transmembrane proteins, it is contemplated thatthese proteins form a secretion apparatus. Some of the proteins in theess gene cluster may actively transport secreted substrates (acting asmotor) while others may regulate transport (regulator). Regulation maybe achieved, but need not be limited to, transcriptional orpost-translational mechanisms for secreted polypeptides, sorting ofspecific substrates to defined locations (e.g., extracellular medium orhost cells), or timing of secretion events during infection. At thispoint, it is unclear whether all secreted Esx proteins function astoxins or contribute indirectly to pathogenesis.

Staphylococci rely on surface protein mediated-adhesion to host cells orinvasion of tissues as a strategy for escape from immune defenses.Furthermore, S. aureus utilize surface proteins to sequester iron fromthe host during infection. The majority of surface proteins involved instaphylococcal pathogenesis carry C-terminal sorting signals, i.e., theyare covalently linked to the cell wall envelope by sortase. Further,staphylococcal strains lacking the genes required for surface proteinanchoring, i.e., sortase A and B, display a dramatic defect in thevirulence in several different mouse models of disease. Thus, surfaceprotein antigens represent a validated vaccine target as thecorresponding genes are essential for the development of staphylococcaldisease and can be exploited in various embodiments of the invention.The sortase enzyme superfamily are Gram-positive transpeptidasesresponsible for anchoring surface protein virulence factors to thepeptidoglycan cell wall layer. Two sortase isoforms have been identifiedin Staphylococcus aureus, SrtA and SrtB. These enzymes have been shownto recognize a LPXTG motif in substrate proteins. The SrtB isoformappears to be important in heme iron acquisition and iron homeostasis,whereas the SrtA isoform plays a critical role in the pathogenesis ofGram-positive bacteria by modulating the ability of the bacterium toadhere to host tissue via the covalent anchoring of adhesins and otherproteins to the cell wall peptidoglycan. In certain embodiments the SpAvariants described herein can be used in combination with otherstaphylococcal proteins such as Coa, Eap, Ebh, Emp, EsaC, EsaB, EsxA,EsxB, Hla, SdrC, SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, IsdC, SasF, vWbp,and/or vWh proteins.

Certain aspects of the invention include methods and compositionsconcerning proteinaceous compositions including polypeptides, peptides,or nucleic acid encoding SpA variant(s) and other staphylococcalantigens such as other proteins transported by the Ess pathway, orsortase substrates. These proteins may be modified by deletion,insertion, and/or substitution.

The Esx polypeptides include the amino acid sequence of Esx proteinsfrom bacteria in the Staphylococcus genus. The Esx sequence may be froma particular staphylococcus species, such as Staphylococcus aureus, andmay be from a particular strain, such as Newman. In certain embodiments,the EsxA sequence is SAV0282 from strain Mu50 (which is the same aminoacid sequence for Newman) and can be accessed using Genbank AccessionNumber Q99WU4 (gi|68565539), which is hereby incorporated by reference.In other embodiments, the EsxB sequence is SAV0290 from strain Mu50(which is the same amino acid sequence for Newman) and can be accessedusing Genbank Accession Number Q99WT7 (gi|68565532), which is herebyincorporated by reference. In further embodiments, other polypeptidestransported by the Ess pathway may be used, the sequences of which maybe identified by one of skill in the art using databases and internetaccessible resources.

The sortase substrate polypeptides include, but are not limited to theamino acid sequence of SdrC, SdrD, SdrE, IsdA, IsdB, ClfA, ClfB, IsdC orSasF proteins from bacteria in the Staphylococcus genus. The sortasesubstrate polypeptide sequence may be from a particular staphylococcusspecies, such as Staphylococcus aureus, and may be from a particularstrain, such as Newman. In certain embodiments, the SdrD sequence isfrom strain N315 and can be accessed using Genbank Accession NumberNP_(—)373773.1 (gi|15926240), which is incorporated by reference. Inother embodiments, the SdrE sequence is from strain N315 and can beaccessed using Genbank Accession Number NP_(—)373774.1 (gi|15926241),which is incorporated by reference. In other embodiments, the IsdAsequence is SAV1130 from strain Mu50 (which is the same amino acidsequence for Newman) and can be accessed using Genbank Accession NumberNP_(—)371654.1 (gi|15924120), which is incorporated by reference. Inother embodiments, the IsdB sequence is SAV1129 from strain Mu50 (whichis the same amino acid sequence for Newman) and can be accessed usingGenbank Accession Number NP_(—)371653.1 (gi|15924119), which isincorporated by reference. In further embodiments, other polypeptidestransported by the Ess pathway or processed by sortase may be used, thesequences of which may be identified by one of skill in the art usingdatabases and internet accessible resources.

Examples of various proteins that can be used in the context of thepresent invention can be identified by analysis of database submissionsof bacterial genomes, including but not limited to accession numbersNC_(—)002951 (GI:57650036 and GenBank CP000046), NC_(—)002758(GI:57634611 and GenBank BA000017), NC_(—)002745 (GI:29165615 andGenBank BA000018), NC_(—)003923 (GI:21281729 and GenBank BA000033),NC_(—)002952 (GI:49482253 and GenBank BX571856), NC_(—)002953(GI:49484912 and GenBank BX571857), NC_(—)007793 (GI:87125858 andGenBank CP000255), NC_(—)007795 (GI:87201381 and GenBank CP000253) eachof which are incorporated by reference.

As used herein, a “protein” or “polypeptide” refers to a moleculecomprising at least ten amino acid residues. In some embodiments, awild-type version of a protein or polypeptide are employed, however, inmany embodiments of the invention, a modified protein or polypeptide isemployed to generate an immune response. The terms described above maybe used interchangeably. A “modified protein” or “modified polypeptide”or a “variant” refers to a protein or polypeptide whose chemicalstructure, particularly its amino acid sequence, is altered with respectto the wild-type protein or polypeptide. In some embodiments, amodified/variant protein or polypeptide has at least one modifiedactivity or function (recognizing that proteins or polypeptides may havemultiple activities or functions). It is specifically contemplated thata modified/variant protein or polypeptide may be altered with respect toone activity or function yet retain a wild-type activity or function inother respects, such as immunogenicity.

In certain embodiments the size of a protein or polypeptide (wild-typeor modified) may comprise, but is not limited to, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240,250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575,600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925,950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500amino molecules or greater, and any range derivable therein, orderivative of a corresponding amino sequence described or referencedherein. It is contemplated that polypeptides may be mutated bytruncation, rendering them shorter than their corresponding wild-typeform, but also they might be altered by fusing or conjugating aheterologous protein sequence with a particular function (e.g., fortargeting or localization, for enhanced immunogenicity, for purificationpurposes, etc.).

As used herein, an “amino molecule” refers to any amino acid, amino acidderivative, or amino acid mimic known in the art. In certainembodiments, the residues of the proteinaceous molecule are sequential,without any non-amino molecule interrupting the sequence of aminomolecule residues. In other embodiments, the sequence may comprise oneor more non-amino molecule moieties. In particular embodiments, thesequence of residues of the proteinaceous molecule may be interrupted byone or more non-amino molecule moieties.

Accordingly, the term “proteinaceous composition” encompasses aminomolecule sequences comprising at least one of the 20 common amino acidsin naturally synthesized proteins, or at least one modified or unusualamino acid.

Proteinaceous compositions may be made by any technique known to thoseof skill in the art, including (i) the expression of proteins,polypeptides, or peptides through standard molecular biologicaltechniques, (ii) the isolation of proteinaceous compounds from naturalsources, or (iii) the chemical synthesis of proteinaceous materials. Thenucleotide as well as the protein, polypeptide, and peptide sequencesfor various genes have been previously disclosed, and may be found inthe recognized computerized databases. One such database is the NationalCenter for Biotechnology Information's Genbank and GenPept databases (onthe World Wide Web at ncbi.nlm.nih.gov/). The coding regions for thesegenes may be amplified and/or expressed using the techniques disclosedherein or as would be known to those of ordinary skill in the art.

Amino acid sequence variants of SpA, coagulases and other polypeptidesof the invention can be substitutional, insertional, or deletionvariants. A variation in a polypeptide of the invention may affect 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more non-contiguous orcontiguous amino acids of the polypeptide, as compared to wild-type. Avariant can comprise an amino acid sequence that is at least 50%, 60%,70%, 80%, or 90%, including all values and ranges there between,identical to any sequence provided or referenced herein, e.g., SEQ IDNO:2-8 or SEQ ID NO:11-30. A variant can include 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more substitute aminoacids. A polypeptide processed or secreted by the Ess pathway or othersurface proteins (see Table 1) or sortase substrates from anystaphylococcus species and strain are contemplated for use incompositions and methods described herein.

Deletion variants typically lack one or more residues of the native orwild-type protein. Individual residues can be deleted or a number ofcontiguous amino acids can be deleted. A stop codon may be introduced(by substitution or insertion) into an encoding nucleic acid sequence togenerate a truncated protein. Insertional mutants typically involve theaddition of material at a non-terminal point in the polypeptide. Thismay include the insertion of one or more residues. Terminal additions,called fusion proteins, may also be generated. These fusion proteinsinclude multimers or concatamers of one or more peptide or polypeptidedescribed or referenced herein.

Substitutional variants typically contain the exchange of one amino acidfor another at one or more sites within the protein, and may be designedto modulate one or more properties of the polypeptide, with or withoutthe loss of other functions or properties. Substitutions may beconservative, that is, one amino acid is replaced with one of similarshape and charge. Conservative substitutions are well known in the artand include, for example, the changes of: alanine to serine; arginine tolysine; asparagine to glutamine or histidine; aspartate to glutamate;cysteine to serine; glutamine to asparagine; glutamate to aspartate;glycine to proline; histidine to asparagine or glutamine; isoleucine toleucine or valine; leucine to valine or isoleucine; lysine to arginine;methionine to leucine or isoleucine; phenylalanine to tyrosine, leucineor methionine; serine to threonine; threonine to serine; tryptophan totyrosine; tyrosine to tryptophan or phenylalanine; and valine toisoleucine or leucine. Alternatively, substitutions may benon-conservative such that a function or activity of the polypeptide isaffected. Non-conservative changes typically involve substituting aresidue with one that is chemically dissimilar, such as a polar orcharged amino acid for a nonpolar or uncharged amino acid, and viceversa.

TABLE 1 Exemplary surface proteins of S. aureus strains. SAV # SA#Surface MW2 Mu50 N315 Newman MRSA252* MSSA476* SAV0111 SA0107 Spa 492450 450 520 516 492 SAV2503 SA2291 FnBPA 1015 1038 1038 741 — 1015SAV2502 SA2290 FnBPB 943 961 961 677 965 957 SAV0811 SA0742 ClfA 946 935989 933 1029 928 SAV2630 SA2423 ClfB 907 877 877 913 873 905 Np Np Cna1183 — — — 1183 1183 SAV0561 SA0519 SdrC 955 953 953 947 906 957 SAV0562SA0520 SdrD 1347 1385 1385 1315 — 1365 SAV0563 SA0521 SdrE 1141 11411141 1166 1137 1141 Np Np Pls — — — — — — SAV2654 SA2447 SasA 2275 22712271 2271 1351 2275 SAV2160 SA1964 SasB 686 2481 2481 2481 2222 685SA1577 SasC 2186 213 2186 2186 2189 2186 SAV0134 SA0129 SasD 241 241 241241 221 241 SAV1130 SA0977 SasE/IsdA 350 350 350 350 354 350 SAV2646SA2439 SasF 635 635 635 635 627 635 SAV2496 SasG 1371 525 927 — — 1371SAV0023 SA0022 SasH 772 — 772 772 786 786 SAV1731 SA1552 SasI 895 891891 891 534 895 SAV1129 SA0976 SasJ/IsdB 645 645 645 645 652 645 SA2381SasK 198 211 211 — — 197 Np SasL — 232 — — — — SAV1131 SA0978 IsdC 227227 227 227 227 227

Proteins of the invention may be recombinant, or synthesized in vitro.Alternatively, a non-recombinant or recombinant protein may be isolatedfrom bacteria. It is also contemplated that a bacteria containing such avariant may be implemented in compositions and methods of the invention.Consequently, a protein need not be isolated.

The term “functionally equivalent codon” is used herein to refer tocodons that encode the same amino acid, such as the six codons forarginine or serine, and also refers to codons that encode biologicallyequivalent amino acids (see Table 2, below).

TABLE 2 Codon Table Amino Acids Codons Alanine Ala A GCA GCC GCG GCUCysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu EGAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGUHistidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys KAAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUGAsparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln QCAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser SAGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val VGUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

It also will be understood that amino acid and nucleic acid sequencesmay include additional residues, such as additional N- or C-terminalamino acids, or 5′ or 3′ sequences, respectively, and yet still beessentially as set forth in one of the sequences disclosed herein, solong as the sequence meets the criteria set forth above, including themaintenance of biological protein activity (e.g., immunogenicity) whereprotein expression is concerned. The addition of terminal sequencesparticularly applies to nucleic acid sequences that may, for example,include various non-coding sequences flanking either of the 5′ or 3′portions of the coding region.

The following is a discussion based upon changing of the amino acids ofa protein to create a variant polypeptide or peptide. For example,certain amino acids may be substituted for other amino acids in aprotein structure with or without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's functional activity, certain amino acid substitutions can bemade in a protein sequence, and in its underlying DNA coding sequence,and nevertheless produce a protein with a desirable property. It is thuscontemplated by the inventors that various changes may be made in theDNA sequences of genes.

It is contemplated that in compositions of the invention, there isbetween about 0.001 mg and about 10 mg of total polypeptide, peptide,and/or protein per ml. The concentration of protein in a composition canbe about, at least about or at most about 0.001, 0.010, 0.050, 0.1, 0.2,0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0,4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 mg/ml ormore (or any range derivable therein). Of this, about, at least about,or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% may be an SpA variantor a coagulase, and may be used in combination with other peptides orpolypeptides, such as other bacterial peptides and/or antigens.

The present invention contemplates the administration of variant SpApolypeptides or peptides to effect a preventative therapy or therapeuticeffect against the development of a disease or condition associated withinfection by a staphylococcus pathogen.

In certain aspects, combinations of staphylococcal antigens are used inthe production of an immunogenic composition that is effective attreating or preventing staphylococcal infection. Staphylococcalinfections progress through several different stages. For example, thestaphylococcal life cycle involves commensal colonization, initiation ofinfection by accessing adjoining tissues or the bloodstream, and/oranaerobic multiplication in the blood. The interplay between S. aureusvirulence determinants and the host defense mechanisms can inducecomplications such as endocarditis, metastatic abscess formation, andsepsis syndrome. Different molecules on the surface of the bacterium areinvolved in different steps of the infection cycle. Combinations ofcertain antigens can elicit an immune response which protects againstmultiple stages of staphylococcal infection. The effectiveness of theimmune response can be measured either in animal model assays and/orusing an opsonophagocytic assay.

D. Polypeptides and Polypeptide Production

The present invention describes polypeptides, peptides, and proteins andimmunogenic fragments thereof for use in various embodiments of thepresent invention. For example, specific polypeptides are assayed for orused to elicit an immune response. In specific embodiments, all or partof the proteins of the invention can also be synthesized in solution oron a solid support in accordance with conventional techniques. Variousautomatic synthesizers are commercially available and can be used inaccordance with known protocols. See, for example, Stewart and Young,(1984); Tam et al., (1983); Merrifield, (1986); and Barany andMerrifield (1979), each incorporated herein by reference.

Alternatively, recombinant DNA technology may be employed wherein anucleotide sequence which encodes a peptide of the invention is insertedinto an expression vector, transformed or transfected into anappropriate host cell and cultivated under conditions suitable forexpression.

One embodiment of the invention includes the use of gene transfer tocells, including microorganisms, for the production and/or presentationof polypeptides or peptides. The gene for the polypeptide or peptide ofinterest may be transferred into appropriate host cells followed byculture of cells under the appropriate conditions. The generation ofrecombinant expression vectors, and the elements included therein, arewell known in the art and briefly discussed herein. Alternatively, theprotein to be produced may be an endogenous protein normally synthesizedby the cell that is isolated and purified.

Another embodiment of the present invention uses autologous B lymphocytecell lines, which are transfected with a viral vector that expresses animmunogen product, and more specifically, a protein having immunogenicactivity. Other examples of mammalian host cell lines include, but arenot limited to Vero and HeLa cells, other B- and T-cell lines, such asCEM, 721.221, H9, Jurkat, Raji, as well as cell lines of Chinese hamsterovary, W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cells. Inaddition, a host cell strain may be chosen that modulates the expressionof the inserted sequences, or that modifies and processes the geneproduct in the manner desired. Such modifications (e.g., glycosylation)and processing (e.g., cleavage) of protein products may be important forthe function of the protein. Different host cells have characteristicand specific mechanisms for the post-translational processing andmodification of proteins. Appropriate cell lines or host systems can bechosen to ensure the correct modification and processing of the foreignprotein expressed.

A number of selection systems may be used including, but not limited toHSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase,and adenine phosphoribosyltransferase genes, in tk-, hgprt- oraprt-cells, respectively. Also, anti-metabolite resistance can be usedas the basis of selection: for dhfr, which confers resistance totrimethoprim and methotrexate; gpt, which confers resistance tomycophenolic acid; neo, which confers resistance to the aminoglycosideG418; and hygro, which confers resistance to hygromycin.

Animal cells can be propagated in vitro in two modes: asnon-anchorage-dependent cells growing in suspension throughout the bulkof the culture or as anchorage-dependent cells requiring attachment to asolid substrate for their propagation (i.e., a monolayer type of cellgrowth).

Non-anchorage dependent or suspension cultures from continuousestablished cell lines are the most widely used means of large scaleproduction of cells and cell products. However, suspension culturedcells have limitations, such as tumorigenic potential and lower proteinproduction than adherent cells.

Where a protein is specifically mentioned herein, it is preferably areference to a native or recombinant protein or optionally a protein inwhich any signal sequence has been removed. The protein may be isolateddirectly from the staphylococcal strain or produced by recombinant DNAtechniques. Immunogenic fragments of the protein may be incorporatedinto the immunogenic composition of the invention. These are fragmentscomprising at least 10 amino acids, 20 amino acids, 30 amino acids, 40amino acids, 50 amino acids, or 100 amino acids, including all valuesand ranges there between, taken contiguously from the amino acidsequence of the protein. In addition, such immunogenic fragments areimmunologically reactive with antibodies generated against theStaphylococcal proteins or with antibodies generated by infection of amammalian host with Staphylococci. Immunogenic fragments also includefragments that when administered at an effective dose, (either alone oras a hapten bound to a carrier), elicit a protective or therapeuticimmune response against Staphylococcal infection, in certain aspects itis protective against S. aureus and/or S. epidermidis infection. Such animmunogenic fragment may include, for example, the protein lacking anN-terminal leader sequence, and/or a transmembrane domain and/or aC-terminal anchor domain. In a preferred aspect the immunogenic fragmentaccording to the invention comprises substantially all of theextracellular domain of a protein which has at least 80% identity, atleast 85% identity, at least 90% identity, at least 95% identity, or atleast 97-99% identity, including all values and ranges there between, toa sequence selected segment of a polypeptide described or referencedherein.

Also included in immunogenic compositions of the invention are fusionproteins composed of one or more Staphylococcal proteins, or immunogenicfragments of staphylococcal proteins. Such fusion proteins may be maderecombinantly and may comprise one portion of at least 1, 2, 3, 4, 5, or6 staphylococcal proteins or segments. Alternatively, a fusion proteinmay comprise multiple portions of at least 1, 2, 3, 4 or 5staphylococcal proteins. These may combine different Staphylococcalproteins and/or multiples of the same protein or proten fragment, orimmunogenic fragments in the same protein (forming a multimer or aconcatamer). Alternatively, the invention also includes individualfusion proteins of Staphylococcal proteins or immunogenic fragmentsthereof, as a fusion protein with heterologous sequences such as aprovider of T-cell epitopes or purification tags, for example:β-galactosidase, glutathione-S-transferase, green fluorescent proteins(GFP), epitope tags such as FLAG, myc tag, poly histidine, or viralsurface proteins such as influenza virus haemagglutinin, or bacterialproteins such as tetanus toxoid, diphtheria toxoid, or CRM197.

Nucleic Acids

In certain embodiments, the present invention concerns recombinantpolynucleotides encoding the proteins, polypeptides, peptides of theinvention. The nucleic acid sequences for SpA, coagulases and otherbacterial proteins are included, all of which are incorporated byreference, and can be used to prepare peptides or polypeptides.

As used in this application, the term “polynucleotide” refers to anucleic acid molecule that either is recombinant or has been isolatedfree of total genomic nucleic acid. Included within the term“polynucleotide” are oligonucleotides (nucleic acids of 100 residues orless in length), recombinant vectors, including, for example, plasmids,cosmids, phage, viruses, and the like. Polynucleotides include, incertain aspects, regulatory sequences, isolated substantially away fromtheir naturally occurring genes or protein encoding sequences.Polynucleotides may be single-stranded (coding or antisense) ordouble-stranded, and may be RNA, DNA (genomic, cDNA or synthetic),analogs thereof, or a combination thereof. Additional coding ornon-coding sequences may, but need not, be present within apolynucleotide.

In this respect, the term “gene,” “polynucleotide,” or “nucleic acid” isused to refer to a nucleic acid that encodes a protein, polypeptide, orpeptide (including any sequences required for proper transcription,post-translational modification, or localization). As will be understoodby those in the art, this term encompasses genomic sequences, expressioncassettes, cDNA sequences, and smaller engineered nucleic acid segmentsthat express, or may be adapted to express, proteins, polypeptides,domains, peptides, fusion proteins, and mutants. A nucleic acid encodingall or part of a polypeptide may contain a contiguous nucleic acidsequence of: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130,140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270,280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410,420, 430, 440, 441, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540,550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680,690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820,830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960,970, 980, 990, 1000, 1010, 1020, 1030, 1040, 1050, 1060, 1070, 1080,1090, 1095, 1100, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, 7000, 7500, 8000, 9000, 10000, or more nucleotides,nucleosides, or base pairs, including all values and rangestherebetween, of a polynucleotide encoding one or more amino acidsequence described or referenced herein. It also is contemplated that aparticular polypeptide may be encoded by nucleic acids containingvariations having slightly different nucleic acid sequences but,nonetheless, encode the same or substantially similar protein (see Table2 above).

In particular embodiments, the invention concerns isolated nucleic acidsegments and recombinant vectors incorporating nucleic acid sequencesthat encode a variant SpA or coagulase. The term “recombinant” may beused in conjunction with a polynucleotide or polypeptide and generallyrefers to a polypeptide or polynucleotide produced and/or manipulated invitro or that is a replication product of such a molecule.

In other embodiments, the invention concerns isolated nucleic acidsegments and recombinant vectors incorporating nucleic acid sequencesthat encode a variant SpA or coagulase polypeptide or peptide togenerate an immune response in a subject. In various embodiments thenucleic acids of the invention may be used in genetic vaccines.

The nucleic acid segments used in the present invention can be combinedwith other nucleic acid sequences, such as promoters, polyadenylationsignals, additional restriction enzyme sites, multiple cloning sites,other coding segments, and the like, such that their overall length mayvary considerably. It is therefore contemplated that a nucleic acidfragment of almost any length may be employed, with the total lengthpreferably being limited by the ease of preparation and use in theintended recombinant nucleic acid protocol. In some cases, a nucleicacid sequence may encode a polypeptide sequence with additionalheterologous coding sequences, for example to allow for purification ofthe polypeptide, transport, secretion, post-translational modification,or for therapeutic benefits such as targeting or efficacy. As discussedabove, a tag or other heterologous polypeptide may be added to themodified polypeptide-encoding sequence, wherein “heterologous” refers toa polypeptide that is not the same as the modified polypeptide.

In certain other embodiments, the invention concerns isolated nucleicacid segments and recombinant vectors that include within their sequencea contiguous nucleic acid sequence from SEQ ID NO:1 (SpA domain D) orSEQ ID NO:3 (SpA) or any other nucleic acid sequences encodingcoagulases or other secreted virulence factors and/or surface proteinsincluding proteins transported by the Ess pathway, processed by sortase,or proteins incorporated herein by reference.

In certain embodiments, the present invention provides polynucleotidevariants having substantial identity to the sequences disclosed herein;those comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,or 99% or higher sequence identity, including all values and rangesthere between, compared to a polynucleotide sequence of this inventionusing the methods described herein (e.g., BLAST analysis using standardparameters).

The invention also contemplates the use of polynucleotides which arecomplementary to all the above described polynucleotides.

E. Vectors

Polypeptides of the invention may be encoded by a nucleic acid moleculecomprised in a vector. The term “vector” is used to refer to a carriernucleic acid molecule into which a heterologous nucleic acid sequencecan be inserted for introduction into a cell where it can be replicatedand expressed. A nucleic acid sequence can be “heterologous,” whichmeans that it is in a context foreign to the cell in which the vector isbeing introduced or to the nucleic acid in which is incorporated, whichincludes a sequence homologous to a sequence in the cell or nucleic acidbut in a position within the host cell or nucleic acid where it isordinarily not found. Vectors include DNAs, RNAs, plasmids, cosmids,viruses (bacteriophage, animal viruses, and plant viruses), andartificial chromosomes (e.g., YACs). One of skill in the art would bewell equipped to construct a vector through standard recombinanttechniques (for example Sambrook et al., 2001; Ausubel et al., 1996,both incorporated herein by reference). In addition to encoding avariant SpA polypeptide the vector can encode other polypeptidesequences such as a one or more other bacterial peptide, a tag, or animmunogenicity enhancing peptide. Useful vectors encoding such fusionproteins include pIN vectors (Inouye et al., 1985), vectors encoding astretch of histidines, and pGEX vectors, for use in generatingglutathione S-transferase (GST) soluble fusion proteins for laterpurification and separation or cleavage.

The term “expression vector” refers to a vector containing a nucleicacid sequence coding for at least part of a gene product capable ofbeing transcribed. In some cases, RNA molecules are then translated intoa protein, polypeptide, or peptide. Expression vectors can contain avariety of “control sequences,” which refer to nucleic acid sequencesnecessary for the transcription and possibly translation of an operablylinked coding sequence in a particular host organism. In addition tocontrol sequences that govern transcription and translation, vectors andexpression vectors may contain nucleic acid sequences that serve otherfunctions as well and are described herein.

1. Promoters and Enhancers

A “promoter” is a control sequence. The promoter is typically a regionof a nucleic acid sequence at which initiation and rate of transcriptionare controlled. It may contain genetic elements at which regulatoryproteins and molecules may bind such as RNA polymerase and othertranscription factors. The phrases “operatively positioned,”“operatively linked,” “under control,” and “under transcriptionalcontrol” mean that a promoter is in a correct functional location and/ororientation in relation to a nucleic acid sequence to controltranscriptional initiation and expression of that sequence. A promotermay or may not be used in conjunction with an “enhancer,” which refersto a cis-acting regulatory sequence involved in the transcriptionalactivation of a nucleic acid sequence.

Naturally, it may be important to employ a promoter and/or enhancer thateffectively directs the expression of the DNA segment in the cell typeor organism chosen for expression. Those of skill in the art ofmolecular biology generally know the use of promoters, enhancers, andcell type combinations for protein expression (see Sambrook et al.,2001, incorporated herein by reference). The promoters employed may beconstitutive, tissue-specific, or inducible and in certain embodimentsmay direct high level expression of the introduced DNA segment underspecified conditions, such as large-scale production of recombinantproteins or peptides.

Various elements/promoters may be employed in the context of the presentinvention to regulate the expression of a gene. Examples of suchinducible elements, which are regions of a nucleic acid sequence thatcan be activated in response to a specific stimulus, include but are notlimited to Immunoglobulin Heavy Chain (Banerji et al., 1983; Gilles etal., 1983; Grosschedl et al., 1985; Atchinson et al., 1986, 1987; Imleret al., 1987; Weinberger et al., 1984; Kiledjian et al., 1988; Porton etal.; 1990), Immunoglobulin Light Chain (Queen et al., 1983; Picard etal., 1984), T Cell Receptor (Luria et al., 1987; Winoto et al., 1989;Redondo et al.; 1990), HLA DQ α and/or DQ β (Sullivan et al., 1987), βInterferon (Goodbourn et al., 1986; Fujita et al., 1987; Goodbourn etal., 1988), Interleukin-2 (Greene et al., 1989), Interleukin-2 Receptor(Greene et al., 1989; Lin et al., 1990), MHC Class II 5 (Koch et al.,1989), MHC Class II HLA-DR^(a) (Sherman et al., 1989), β-Actin (Kawamotoet al., 1988; Ng et al.; 1989), Muscle Creatine Kinase (MCK) (Jaynes etal., 1988; Horlick et al., 1989; Johnson et al., 1989), Prealbumin(Transthyretin) (Costa et al., 1988), Elastase I (Ornitz et al., 1987),Metallothionein (MTII) (Karin et al., 1987; Culotta et al., 1989),Collagenase (Pinkert et al., 1987; Angel et al., 1987), Albumin (Pinkertet al., 1987; Tronche et al., 1989, 1990), α-Fetoprotein (Godbout etal., 1988; Campere et al., 1989), γ-Globin (Bodine et al., 1987;Perez-Stable et al., 1990), β-Globin (Trudel et al., 1987), c-fos (Cohenet al., 1987), c-Ha-Ras (Triesman, 1986; Deschamps et al., 1985),Insulin (Edlund et al., 1985), Neural Cell Adhesion Molecule (NCAM)(Hirsh et al., 1990), α1-Antitrypain (Latimer et al., 1990), H₂B (TH2B)Histone (Hwang et al., 1990), Mouse and/or Type I Collagen (Ripe et al.,1989), Glucose-Regulated Proteins (GRP94 and GRP78) (Chang et al.,1989), Rat Growth Hormone (Larsen et al., 1986), Human Serum Amyloid A(SAA) (Edbrooke et al., 1989), Troponin I (TN I) (Yutzey et al., 1989),Platelet-Derived Growth Factor (PDGF) (Pech et al., 1989), DuchenneMuscular Dystrophy (Klamut et al., 1990), SV40 (Banerji et al., 1981;Moreau et al., 1981; Sleigh et al., 1985; Firak et al., 1986; Herr etal., 1986; Imbra et al., 1986; Kadesch et al., 1986; Wang et al., 1986;Ondek et al., 1987; Kuhl et al., 1987; Schaffner et al., 1988), Polyoma(Swartzendruber et al., 1975; Vasseur et al., 1980; Katinka et al.,1980, 1981; Tyndell et al., 1981; Dandolo et al., 1983; de Villiers etal., 1984; Hen et al., 1986; Satake et al., 1988; Campbell et al.,1988), Retroviruses (Kriegler et al., 1982, 1983; Levinson et al., 1982;Kriegler et al., 1983, 1984a, b, 1988; Bosze et al., 1986; Miksicek etal., 1986; Celander et al., 1987; Thiesen et al., 1988; Celander et al.,1988; Choi et al., 1988; Reisman et al., 1989), Papilloma Virus (Campoet al., 1983; Lusky et al., 1983; Spandidos and Wilkie, 1983; Spalholzet al., 1985; Lusky et al., 1986; Cripe et al., 1987; Gloss et al.,1987; Hirochika et al., 1987; Stephens et al., 1987), Hepatitis B Virus(Bulla et al., 1986; Jameel et al., 1986; Shaul et al., 1987; Spandau etal., 1988; Vannice et al., 1988), Human Immunodeficiency Virus (Muesinget al., 1987; Hauber et al., 1988; Jakobovits et al., 1988; Feng et al.,1988; Takebe et al., 1988; Rosen et al., 1988; Berkhout et al., 1989;Laspia et al., 1989; Sharp et al., 1989; Braddock et al., 1989),Cytomegalovirus (CMV) IE (Weber et al., 1984; Boshart et al., 1985;Foecking et al., 1986), Gibbon Ape Leukemia Virus (Holbrook et al.,1987; Quinn et al., 1989).

Inducible elements include, but are not limited to MT II—Phorbol Ester(TFA)/Heavy metals (Palmiter et al., 1982; Haslinger et al., 1985;Searle et al., 1985; Stuart et al., 1985; Imagawa et al., 1987, Karin etal., 1987; Angel et al., 1987b; McNeall et al., 1989); MMTV (mousemammary tumor virus)—Glucocorticoids (Huang et al., 1981; Lee et al.,1981; Majors et al., 1983; Chandler et al., 1983; Lee et al., 1984;Ponta et al., 1985; Sakai et al., 1988); β-Interferon—poly(rI)x/poly(rc)(Tavernier et al., 1983); Adenovirus 5 E2-E1A (Imperiale et al., 1984);Collagenase—Phorbol Ester (TPA) (Angel et al., 1987a);Stromelysin—Phorbol Ester (TPA) (Angel et al., 1987b); SV40—PhorbolEster (TPA) (Angel et al., 1987b); Murine MX Gene—Interferon, NewcastleDisease Virus (Hug et al., 1988); GRP78 Gene—A23187 (Resendez et al.,1988); α-2-Macroglobulin—IL-6 (Kunz et al., 1989); Vimentin—Serum(Rittling et al., 1989); MHC Class I Gene H-2κb—Interferon (Blanar etal., 1989); HSP70—E1A/SV40 Large T Antigen (Taylor et al., 1989, 1990a,1990b); Proliferin—Phorbol Ester/TPA (Mordacq et al., 1989); TumorNecrosis Factor—PMA (Hensel et al., 1989); and Thyroid StimulatingHormone a Gene—Thyroid Hormone (Chatterjee et al., 1989).

The particular promoter that is employed to control the expression ofpeptide or protein encoding polynucleotide of the invention is notbelieved to be critical, so long as it is capable of expressing thepolynucleotide in a targeted cell, preferably a bacterial cell. Where ahuman cell is targeted, it is preferable to position the polynucleotidecoding region adjacent to and under the control of a promoter that iscapable of being expressed in a human cell. Generally speaking, such apromoter might include either a bacterial, human or viral promoter.

In embodiments in which a vector is administered to a subject forexpression of the protein, it is contemplated that a desirable promoterfor use with the vector is one that is not down-regulated by cytokinesor one that is strong enough that even if down-regulated, it produces aneffective amount of a variant SpA for eliciting an immune response.Non-limiting examples of these are CMV IE and RSV LTR. Tissue specificpromoters can be used, particularly if expression is in cells in whichexpression of an antigen is desirable, such as dendritic cells ormacrophages. The mammalian MHC I and MHC II promoters are examples ofsuch tissue-specific promoters.

2. Initiation Signals and Internal Ribosome Binding Sites (IRES)

A specific initiation signal also may be required for efficienttranslation of coding sequences. These signals include the ATGinitiation codon or adjacent sequences. Exogenous translational controlsignals, including the ATG initiation codon, may need to be provided.One of ordinary skill in the art would readily be capable of determiningthis and providing the necessary signals.

In certain embodiments of the invention, the use of internal ribosomeentry sites (IRES) elements are used to create multigene, orpolycistronic, messages. IRES elements are able to bypass the ribosomescanning model of 5{tilde over (′)} methylated Cap dependent translationand begin translation at internal sites (Pelletier and Sonenberg, 1988;Macejak and Sarnow, 1991). IRES elements can be linked to heterologousopen reading frames. Multiple open reading frames can be transcribedtogether, each separated by an IRES, creating polycistronic messages.Multiple genes can be efficiently expressed using a singlepromoter/enhancer to transcribe a single message (see U.S. Pat. Nos.5,925,565 and 5,935,819, herein incorporated by reference).

3. Selectable and Screenable Markers

In certain embodiments of the invention, cells containing a nucleic acidconstruct of the present invention may be identified in vitro or in vivoby encoding a screenable or selectable marker in the expression vector.When transcribed and translated, a marker confers an identifiable changeto the cell permitting easy identification of cells containing theexpression vector. Generally, a selectable marker is one that confers aproperty that allows for selection. A positive selectable marker is onein which the presence of the marker allows for its selection, while anegative selectable marker is one in which its presence prevents itsselection. An example of a positive selectable marker is a drugresistance marker.

F. Host Cells

As used herein, the terms “cell,” “cell line,” and “cell culture” may beused interchangeably. All of these terms also include their progeny,which is any and all subsequent generations. It is understood that allprogeny may not be identical due to deliberate or inadvertent mutations.In the context of expressing a heterologous nucleic acid sequence, “hostcell” refers to a prokaryotic or eukaryotic cell, and it includes anytransformable organism that is capable of replicating a vector orexpressing a heterologous gene encoded by a vector. A host cell can, andhas been, used as a recipient for vectors or viruses. A host cell may be“transfected” or “transformed,” which refers to a process by whichexogenous nucleic acid, such as a recombinant protein-encoding sequence,is transferred or introduced into the host cell. A transformed cellincludes the primary subject cell and its progeny.

Host cells may be derived from prokaryotes or eukaryotes, includingbacteria, yeast cells, insect cells, and mammalian cells for replicationof the vector or expression of part or all of the nucleic acidsequence(s). Numerous cell lines and cultures are available for use as ahost cell, and they can be obtained through the American Type CultureCollection (ATCC), which is an organization that serves as an archivefor living cultures and genetic materials (www.atcc.org).

G. Expression Systems

Numerous expression systems exist that comprise at least a part or allof the compositions discussed above. Prokaryote- and/or eukaryote-basedsystems can be employed for use with the present invention to producenucleic acid sequences, or their cognate polypeptides, proteins andpeptides. Many such systems are commercially and widely available.

The insect cell/baculovirus system can produce a high level of proteinexpression of a heterologous nucleic acid segment, such as described inU.S. Pat. Nos. 5,871,986, 4,879,236, both herein incorporated byreference, and which can be bought, for example, under the name MAXBAC®2.0 from INVITROGEN® and BACPACK™ BACULOVIRUS EXPRESSION SYSTEM FROMCLONTECH®.

In addition to the disclosed expression systems of the invention, otherexamples of expression systems include STRATAGENE®'s COMPLETE CONTROL™Inducible Mammalian Expression System, which involves a syntheticecdysone-inducible receptor, or its pET Expression System, an E. coliexpression system. Another example of an inducible expression system isavailable from INVITROGEN®, which carries the T-REX™(tetracycline-regulated expression) System, an inducible mammalianexpression system that uses the full-length CMV promoter. INVITROGEN®also provides a yeast expression system called the Pichia methanolicaExpression System, which is designed for high-level production ofrecombinant proteins in the methylotrophic yeast Pichia methanolica. Oneof skill in the art would know how to express a vector, such as anexpression construct, to produce a nucleic acid sequence or its cognatepolypeptide, protein, or peptide.

V. Polysaccharides

The immunogenic compositions of the invention may further comprisecapsular polysaccharides including one or more of PIA (also known asPNAG) and/or S. aureus Type V and/or type VIII capsular polysaccharideand/or S. epidermidis Type I, and/or Type II and/or Type III capsularpolysaccharide.

H. PIA (PNAG)

It is now clear that the various forms of staphylococcal surfacepolysaccharides identified as PS/A, PIA and SAA are the same chemicalentity—PNAG (Maira-Litran et al., 2004). Therefore the term PIA or PNAGencompasses all these polysaccharides or oligosaccharides derived fromthem.

PIA is a polysaccharide intercellular adhesin and is composed of apolymer of β-(1→6)-linked glucosamine substituted with N-acetyl andO-succinyl constituents. This polysaccharide is present in both S.aureus and S. epidermidis and can be isolated from either source (Joyceet al., 2003; Maira-Litran et al., 2002). For example, PNAG may beisolated from S. aureus strain MN8m (WO04/43407). PIA isolated from S.epidermidis is a integral constituent of biofilm. It is responsible formediating cell-cell adhesion and probably also functions to shield thegrowing colony from the host's immune response. The polysaccharidepreviously known as poly-N-succinyl-β-(1→6)-glucosamine (PNSG) wasrecently shown not to have the expected structure since theidentification of N-succinylation was incorrect (Maira-Litran et al.,2002). Therefore the polysaccharide formally known as PNSG and now foundto be PNAG is also encompassed by the term PIA.

PIA (or PNAG) may be of different sizes varying from over 400 kDa tobetween 75 and 400 kDa to between 10 and 75 kDa to oligosaccharidescomposed of up to 30 repeat units (of β-(1→6)-linked glucosaminesubstituted with N-acetyl and O-succinyl constituents). Any size of PIApolysaccharide or oligosaccharide may be use in an immunogeniccomposition of the invention, in one aspect the polysaccharide is over40 kDa. Sizing may be achieved by any method known in the art, forinstance by microfluidization, ultrasonic irradiation or by chemicalcleavage (WO 03/53462, EP497524, EP497525). In certain aspects PIA(PNAG) is at least or at most 40-400 kDa, 40-300 kDa, 50-350 kDa, 60-300kDa, 50-250 kDa and 60-200 kDa.

PIA (PNAG) can have different degree of acetylation due to substitutionon the amino groups by acetate. PIA produced in vitro is almost fullysubstituted on amino groups (95-100%). Alternatively, a deacetylated PIA(PNAG) can be used having less than 60%, 50%, 40%, 30%, 20%, 10%acetylation. Use of a deacetylated PIA (PNAG) is preferred sincenon-acetylated epitopes of PNAG are efficient at mediating opsonickilling of Gram positive bacteria, preferably S. aureus and/or S.epidermidis. In certain aspects, the PIA (PNAG) has a size between 40kDa and 300 kDa and is deacetylated so that less than 60%, 50%, 40%, 30%or 20% of amino groups are acetylated.

The term deacetylated PNAG (dPNAG) refers to a PNAG polysaccharide oroligosaccharide in which less than 60%, 50%, 40%, 30%, 20% or 10% of theamino agroups are acetylated. In certain aspects, PNAG is deaceylated toform dPNAG by chemically treating the native polysaccharide. Forexample, the native PNAG is treated with a basic solution such that thepH rises to above 10. For instance the PNAG is treated with 0.1-5 M,0.2-4 M, 0.3-3 M, 0.5-2 M, 0.75-1.5 M or 1 M NaOH, KOH or NH₄OH.Treatment is for at least 10 to 30 minutes, or 1, 2, 3, 4, 5, 10, 15 or20 hours at a temperature of 20-100, 25-80, 30-60 or 30-50 or 35-45° C.dPNAG may be prepared as described in WO 04/43405.

The polysaccharide(s) can be conjugated or unconjugated to a carrierprotein.

I. Type 5 and Type 8 Polysaccharides from S. aureus

Most strains of S. aureus that cause infection in man contain eitherType 5 or Type 8 polysaccharides. Approximately 60% of human strains areType 8 and approximately 30% are Type 5. The structures of Type 5 andType 8 capsular polysaccharide antigens are described in Moreau et al.,(1990) and Fournier et al., (1984). Both have FucNAcp in their repeatunit as well as ManNAcA which can be used to introduce a sulfhydrylgroup. The structures are:

Type 5

→4)-β-D-ManNAcA(3OAc)-(1→4)-α-L-FucNAc(1→3)-β-D-FucNAc-(1→

Type 8

→3)-β-D-ManNAcA(4OAc)-(1→3)-α-L-FucNAc(1→3)-β-D-FucNAc-(1→

Recently (Jones, 2005) NMR spectroscopy revised the structures to:

Type 5

→4)-β-D-ManNAcA-(1→4)-α-L-FucNAc(3OAc)-(1→3)-β-D-FucNAc-(1→

Type 8

→3)-β-D-ManNAcA(4OAc)-(1→3)-α-L-FucNAc(1→3)-α-D-FucNAc(1→

Polysaccharides may be extracted from the appropriate strain of S.aureus using method well known to of skill in the art, See U.S. Pat. No.6,294,177. For example, ATCC 12902 is a Type 5 S. aureus strain and ATCC12605 is a Type 8 S. aureus strain.

Polysaccharides are of native size or alternatively may be sized, forinstance by microfluidisation, ultrasonic irradiation, or by chemicaltreatment. The invention also covers oligosaccharides derived from thetype 5 and 8 polysaccharides from S. aureus. The type 5 and 8polysaccharides included in the immunogenic composition of the inventionare preferably conjugated to a carrier protein as described below or arealternatively unconjugated. The immunogenic compositions of theinvention alternatively contains either type 5 or type 8 polysaccharide.

J. S. aureus 336 antigen

In an embodiment, the immunogenic composition of the invention comprisesthe S. aureus 336 antigen described in U.S. Pat. No. 6,294,177. The 336antigen comprises β-linked hexosamine, contains no O-acetyl groups, andspecifically binds to antibodies to S. aureus Type 336 deposited underATCC 55804. In an embodiment, the 336 antigen is a polysaccharide whichis of native size or alternatively may be sized, for instance bymicrofluidisation, ultrasonic irradiation, or by chemical treatment. Theinvention also covers oligosaccharides derived from the 336 antigen. The336 antigen can be unconjugated or conjugated to a carrier protein.

K. Type I, II and III polysaccharides from S. epidermidis

Amongst the problems associated with the use of polysaccharides invaccination, is the fact that polysaccharides per se are poorimmunogens. It is preferred that the polysaccharides utilized in theinvention are linked to a protein carrier which provide bystander T-cellhelp to improve immunogenicity. Examples of such carriers which may beconjugated to polysaccharide immunogens include the Diphtheria andTetanus toxoids (DT, DT CRM197 and TT respectively), Keyhole LimpetHaemocyanin (KLH), and the purified protein derivative of Tuberculin(PPD), Pseudomonas aeruginosa exoprotein A (rEPA), protein D fromHaemophilus influenzae, pneumolysin or fragments of any of the above.Fragments suitable for use include fragments encompassing T-helperepitopes. In particular the protein D fragment from H. influenza willpreferably contain the N-terminal ⅓ of the protein. Protein D is anIgD-binding protein from Haemophilus influenzae (EP 0 594 610 B1) and isa potential immunogen. In addition, staphylococcal proteins may be usedas a carrier protein in the polysaccharide conjugates of the invention.

A carrier protein that would be particularly advantageous to use in thecontext of a staphylococcal vaccine is staphylococcal alpha toxoid. Thenative form may be conjugated to a polysaccharide since the process ofconjugation reduces toxicity. Preferably genetically detoxified alphatoxins such as the His35Leu or His35Arg variants are used as carrierssince residual toxicity is lower. Alternatively the alpha toxin ischemically detoxified by treatment with a cross-linking reagent,formaldehyde or glutaraldehyde. A genetically detoxified alpha toxin isoptionally chemically detoxified, preferably by treatment with across-linking reagent, formaldehyde or glutaraldehyde to further reducetoxicity.

The polysaccharides may be linked to the carrier protein(s) by any knownmethod (for example those methods described in U.S. Pat. Nos. 4,372,945,4,474,757, and 4,356,170). Preferably, CDAP conjugation chemistry iscarried out (see WO95/08348). In CDAP, the cyanylating reagent1-cyano-dimethylaminopyridinium tetrafluoroborate (CDAP) is preferablyused for the synthesis of polysaccharide-protein conjugates. Thecyanilation reaction can be performed under relatively mild conditions,which avoids hydrolysis of the alkaline sensitive polysaccharides. Thissynthesis allows direct coupling to a carrier protein.

Conjugation preferably involves producing a direct linkage between thecarrier protein and polysaccharide. Optionally a spacer (such as adipicdihydride (ADH)) may be introduced between the carrier protein and thepolysaccharide.

IV. Immune Response and Assays

As discussed above, the invention concerns evoking or inducing an immuneresponse in a subject against a variant SpA or coagulase peptide. In oneembodiment, the immune response can protect against or treat a subjecthaving, suspected of having, or at risk of developing an infection orrelated disease, particularly those related to staphylococci. One use ofthe immunogenic compositions of the invention is to prevent nosocomialinfections by inoculating a subject prior to undergoing procedures in ahospital or other environment having an increased risk of infection.

A. Immunoassays

The present invention includes the implementation of serological assaysto evaluate whether and to what extent an immune response is induced orevoked by compositions of the invention. There are many types ofimmunoassays that can be implemented. Immunoassays encompassed by thepresent invention include, but are not limited to, those described inU.S. Pat. No. 4,367,110 (double monoclonal antibody sandwich assay) andU.S. Pat. No. 4,452,901 (western blot). Other assays includeimmunoprecipitation of labeled ligands and immunocytochemistry, both invitro and in vivo.

Immunoassays generally are binding assays. Certain preferredimmunoassays are the various types of enzyme linked immunosorbent assays(ELISAs) and radioimmunoassays (RIA) known in the art.Immunohistochemical detection using tissue sections is also particularlyuseful. In one example, antibodies or antigens are immobilized on aselected surface, such as a well in a polystyrene microtiter plate,dipstick, or column support. Then, a test composition suspected ofcontaining the desired antigen or antibody, such as a clinical sample,is added to the wells. After binding and washing to remove nonspecifically bound immune complexes, the bound antigen or antibody maybe detected. Detection is generally achieved by the addition of anotherantibody, specific for the desired antigen or antibody, that is linkedto a detectable label. This type of ELISA is known as a “sandwichELISA.” Detection also may be achieved by the addition of a secondantibody specific for the desired antigen, followed by the addition of athird antibody that has binding affinity for the second antibody, withthe third antibody being linked to a detectable label.

Competition ELISAs are also possible implementations in which testsamples compete for binding with known amounts of labeled antigens orantibodies. The amount of reactive species in the unknown sample isdetermined by mixing the sample with the known labeled species before orduring incubation with coated wells. The presence of reactive species inthe sample acts to reduce the amount of labeled species available forbinding to the well and thus reduces the ultimate signal. Irrespectiveof the format employed, ELISAs have certain features in common, such ascoating, incubating or binding, washing to remove non specifically boundspecies, and detecting the bound immune complexes.

Antigen or antibodies may also be linked to a solid support, such as inthe form of plate, beads, dipstick, membrane, or column matrix, and thesample to be analyzed is applied to the immobilized antigen or antibody.In coating a plate with either antigen or antibody, one will generallyincubate the wells of the plate with a solution of the antigen orantibody, either overnight or for a specified period. The wells of theplate will then be washed to remove incompletely-adsorbed material. Anyremaining available surfaces of the wells are then “coated” with anonspecific protein that is antigenically neutral with regard to thetest antisera. These include bovine serum albumin (BSA), casein, andsolutions of milk powder. The coating allows for blocking of nonspecificadsorption sites on the immobilizing surface and thus reduces thebackground caused by nonspecific binding of antisera onto the surface.

B. Diagnosis of Bacterial Infection

In addition to the use of proteins, polypeptides, and/or peptides, aswell as antibodies binding these polypeptides, proteins, and/orpeptides, to treat or prevent infection as described above, the presentinvention contemplates the use of these polypeptides, proteins,peptides, and/or antibodies in a variety of ways, including thedetection of the presence of Staphylococci to diagnose an infection,whether in a patient or on medical equipment which may also becomeinfected. In accordance with the invention, a preferred method ofdetecting the presence of infections involves the steps of obtaining asample suspected of being infected by one or more staphylococcalbacteria species or strains, such as a sample taken from an individual,for example, from one's blood, saliva, tissues, bone, muscle, cartilage,or skin. Following isolation of the sample, diagnostic assays utilizingthe polypeptides, proteins, peptides, and/or antibodies of the presentinvention may be carried out to detect the presence of staphylococci,and such assay techniques for determining such presence in a sample arewell known to those skilled in the art and include methods such asradioimmunoassay, western blot analysis and ELISA assays. In general, inaccordance with the invention, a method of diagnosing an infection iscontemplated wherein a sample suspected of being infected withstaphylococci has added to it the polypeptide, protein, peptide,antibody, or monoclonal antibody in accordance with the presentinvention, and staphylococci are indicated by antibody binding to thepolypeptides, proteins, and/or peptides, or polypeptides, proteins,and/or peptides binding to the antibodies in the sample.

Accordingly, antibodies in accordance with the invention may be used forthe prevention of infection from staphylococcal bacteria (i.e., passiveimmunization), for the treatment of an ongoing infection, or for use asresearch tools. The term “antibodies” as used herein includesmonoclonal, polyclonal, chimeric, single chain, bispecific, simianized,and humanized or primatized antibodies as well as Fab fragments, such asthose fragments which maintain the binding specificity of theantibodies, including the products of an Fab immunoglobulin expressionlibrary. Accordingly, the invention contemplates the use of singlechains such as the variable heavy and light chains of the antibodies.Generation of any of these types of antibodies or antibody fragments iswell known to those skilled in the art. Specific examples of thegeneration of an antibody to a bacterial protein can be found in U.S.Patent Application Pub. No. 20030153022, which is incorporated herein byreference in its entirety.

Any of the above described polypeptides, proteins, peptides, and/orantibodies may be labeled directly with a detectable label foridentification and quantification of staphylococcal bacteria. Labels foruse in immunoassays are generally known to those skilled in the art andinclude enzymes, radioisotopes, and fluorescent, luminescent andchromogenic substances, including colored particles such as colloidalgold or latex beads. Suitable immunoassays include enzyme-linkedimmunosorbent assays (ELISA).

C. Protective Immunity

In some embodiments of the invention, proteinaceous compositions conferprotective immunity to a subject. Protective immunity refers to a body'sability to mount a specific immune response that protects the subjectfrom developing a particular disease or condition that involves theagent against which there is an immune response. An immunogenicallyeffective amount is capable of conferring protective immunity to thesubject.

As used herein in the specification and in the claims section thatfollows, the term polypeptide or peptide refer to a stretch of aminoacids covalently linked there amongst via peptide bonds. Differentpolypeptides have different functionalities according to the presentinvention. While according to one aspect, a polypeptide is derived froman immunogen designed to induce an active immune response in arecipient, according to another aspect of the invention, a polypeptideis derived from an antibody which results following the elicitation ofan active immune response in, for example, an animal, and which canserve to induce a passive immune response in the recipient. In bothcases, however, the polypeptide is encoded by a polynucleotide accordingto any possible codon usage.

As used herein the phrase “immune response” or its equivalent“immunological response” refers to the development of a humoral(antibody mediated), cellular (mediated by antigen-specific T cells ortheir secretion products) or both humoral and cellular response directedagainst a protein, peptide, carbohydrate, or polypeptide of theinvention in a recipient patient. Such a response can be an activeresponse induced by administration of immunogen or a passive responseinduced by administration of antibody, antibody containing material, orprimed T-cells. A cellular immune response is elicited by thepresentation of polypeptide epitopes in association with Class I orClass II MHC molecules, to activate antigen-specific CD4 (+) T helpercells and/or CD8 (+) cytotoxic T cells. The response may also involveactivation of monocytes, macrophages, NK cells, basophils, dendriticcells, astrocytes, microglia cells, eosinophils or other components ofinnate immunity. As used herein “active immunity” refers to any immunityconferred upon a subject by administration of an antigen.

As used herein “passive immunity” refers to any immunity conferred upona subject without administration of an antigen to the subject. “Passiveimmunity” therefore includes, but is not limited to, administration ofactivated immune effectors including cellular mediators or proteinmediators (e.g., monoclonal and/or polyclonal antibodies) of an immuneresponse. A monoclonal or polyclonal antibody composition may be used inpassive immunization for the prevention or treatment of infection byorganisms that carry the antigen recognized by the antibody. An antibodycomposition may include antibodies that bind to a variety of antigensthat may in turn be associated with various organisms. The antibodycomponent can be a polyclonal antiserum. In certain aspects the antibodyor antibodies are affinity purified from an animal or second subjectthat has been challenged with an antigen(s). Alternatively, an antibodymixture may be used, which is a mixture of monoclonal and/or polyclonalantibodies to antigens present in the same, related, or differentmicrobes or organisms, such as gram-positive bacteria, gram-negativebacteria, including but not limited to staphylococcus bacteria.

Passive immunity may be imparted to a patient or subject byadministering to the patient immunoglobulins (Ig) and/or other immunefactors obtained from a donor or other non-patient source having a knownimmunoreactivity. In other aspects, an antigenic composition of thepresent invention can be administered to a subject who then acts as asource or donor for globulin, produced in response to challenge with theantigenic composition (“hyperimmune globulin”), that contains antibodiesdirected against Staphylococcus or other organism. A subject thustreated would donate plasma from which hyperimmune globulin would thenbe obtained, via conventional plasma-fractionation methodology, andadministered to another subject in order to impart resistance against orto treat staphylococcus infection. Hyperimmune globulins according tothe invention are particularly useful for immune-compromisedindividuals, for individuals undergoing invasive procedures or wheretime does not permit the individual to produce their own antibodies inresponse to vaccination. See U.S. Pat. Nos. 6,936,258, 6,770,278,6,756,361, 5,548,066, 5,512,282, 4,338,298, and 4,748,018, each of whichis incorporated herein by reference in its entirety, for exemplarymethods and compositions related to passive immunity.

For purposes of this specification and the accompanying claims the terms“epitope” and “antigenic determinant” are used interchangeably to referto a site on an antigen to which B and/or T cells respond or recognizeB-cell epitopes can be formed both from contiguous amino acids ornoncontiguous amino acids juxtaposed by tertiary folding of a protein.Epitopes formed from contiguous amino acids are typically retained onexposure to denaturing solvents whereas epitopes formed by tertiaryfolding are typically lost on treatment with denaturing solvents. Anepitope typically includes at least 3, and more usually, at least 5 or8-10 amino acids in a unique spatial conformation. Methods ofdetermining spatial conformation of epitopes include, for example, x-raycrystallography and 2-dimensional nuclear magnetic resonance. See, e.g.,Epitope Mapping Protocols (1996). Antibodies that recognize the sameepitope can be identified in a simple immunoassay showing the ability ofone antibody to block the binding of another antibody to a targetantigen. T-cells recognize continuous epitopes of about nine amino acidsfor CD8 cells or about 13-15 amino acids for CD4 cells. T cells thatrecognize the epitope can be identified by in vitro assays that measureantigen-dependent proliferation, as determined by ³H-thymidineincorporation by primed T cells in response to an epitope (Burke et al.,1994), by antigen-dependent killing (cytotoxic T lymphocyte assay,Tigges et al., 1996) or by cytokine secretion.

The presence of a cell-mediated immunological response can be determinedby proliferation assays (CD4 (+) T cells) or CTL (cytotoxic Tlymphocyte) assays. The relative contributions of humoral and cellularresponses to the protective or therapeutic effect of an immunogen can bedistinguished by separately isolating IgG and T-cells from an immunizedsyngeneic animal and measuring protective or therapeutic effect in asecond subject.

As used herein and in the claims, the terms “antibody” or“immunoglobulin” are used interchangeably and refer to any of severalclasses of structurally related proteins that function as part of theimmune response of an animal or recipient, which proteins include IgG,IgD, IgE, IgA, IgM and related proteins.

Under normal physiological conditions antibodies are found in plasma andother body fluids and in the membrane of certain cells and are producedby lymphocytes of the type denoted B cells or their functionalequivalent. Antibodies of the IgG class are made up of four polypeptidechains linked together by disulfide bonds. The four chains of intact IgGmolecules are two identical heavy chains referred to as H-chains and twoidentical light chains referred to as L-chains.

In order to produce polyclonal antibodies, a host, such as a rabbit orgoat, is immunized with the antigen or antigen fragment, generally withan adjuvant and, if necessary, coupled to a carrier. Antibodies to theantigen are subsequently collected from the sera of the host. Thepolyclonal antibody can be affinity purified against the antigenrendering it monospecific.

Monoclonal antibodies can be produced by hyperimmunization of anappropriate donor with the antigen or ex-vivo by use of primary culturesof splenic cells or cell lines derived from spleen (Anavi, 1998; Hustonet al., 1991; Johnson et al., 1991; Mernaugh et al., 1995).

As used herein and in the claims, the phrase “an immunological portionof an antibody” includes a Fab fragment of an antibody, a Fv fragment ofan antibody, a heavy chain of an antibody, a light chain of an antibody,a heterodimer consisting of a heavy chain and a light chain of anantibody, a variable fragment of a light chain of an antibody, avariable fragment of a heavy chain of an antibody, and a single chainvariant of an antibody, which is also known as scFv. In addition, theterm includes chimeric immunoglobulins which are the expression productsof fused genes derived from different species, one of the species can bea human, in which case a chimeric immunoglobulin is said to behumanized. Typically, an immunological portion of an antibody competeswith the intact antibody from which it was derived for specific bindingto an antigen.

Optionally, an antibody or preferably an immunological portion of anantibody, can be chemically conjugated to, or expressed as, a fusionprotein with other proteins. For purposes of this specification and theaccompanying claims, all such fused proteins are included in thedefinition of antibodies or an immunological portion of an antibody.

As used herein the terms “immunogenic agent” or “immunogen” or “antigen”are used interchangeably to describe a molecule capable of inducing animmunological response against itself on administration to a recipient,either alone, in conjunction with an adjuvant, or presented on a displayvehicle.

D. Treatment Methods

A method of the present invention includes treatment for a disease orcondition caused by a staphylococcus pathogen. An immunogenicpolypeptide of the invention can be given to induce an immune responsein a person infected with staphylococcus or suspected of having beenexposed to staphylococcus. Methods may be employed with respect toindividuals who have tested positive for exposure to staphylococcus orwho are deemed to be at risk for infection based on possible exposure.

In particular, the invention encompasses a method of treatment forstaphylococcal infection, particularly hospital acquired nosocomialinfections. The immunogenic compositions and vaccines of the inventionare particularly advantageous to use in cases of elective surgery. Suchpatients will know the date of surgery in advance and could beinoculated in advance. The immunogenic compositions and vaccines of theinvention are also advantageous to use to inoculate health care workers.

In some embodiments, the treatment is administered in the presence ofadjuvants or carriers or other staphylococcal antigens. Furthermore, insome examples, treatment comprises administration of other agentscommonly used against bacterial infection, such as one or moreantibiotics.

The use of peptides for vaccination can require, but not necessarily,conjugation of the peptide to an immunogenic carrier protein, such ashepatitis B surface antigen, keyhole limpet hemocyanin, or bovine serumalbumin. Methods for performing this conjugation are well known in theart.

VI. Vaccine and other Pharmaceutical Compositions and Administration

E. Vaccines

The present invention includes methods for preventing or amelioratingstaphylococcal infections, particularly hospital acquired nosocomialinfections. As such, the invention contemplates vaccines for use in bothactive and passive immunization embodiments. Immunogenic compositions,proposed to be suitable for use as a vaccine, may be prepared fromimmunogenic SpA polypeptide(s), such as a SpA domain D variant, orimmunogenic coagulases. In other embodiments SpA or coagulases can beused in combination with other secreted virulence proteins, surfaceproteins or immunogenic fragments thereof. In certain aspects, antigenicmaterial is extensively dialyzed to remove undesired small molecularweight molecules and/or lyophilized for more ready formulation into adesired vehicle.

Other options for a protein/peptide-based vaccine involve introducingnucleic acids encoding the antigen(s) as DNA vaccines. In this regard,recent reports described construction of recombinant vaccinia virusesexpressing either 10 contiguous minimal CTL epitopes (Thomson, 1996) ora combination of B cell, cytotoxic T-lymphocyte (CTL), and T-helper (Th)epitopes from several microbes (An, 1997), and successful use of suchconstructs to immunize mice for priming protective immune responses.Thus, there is ample evidence in the literature for successfulutilization of peptides, peptide-pulsed antigen presenting cells (APCs),and peptide-encoding constructs for efficient in vivo priming ofprotective immune responses. The use of nucleic acid sequences asvaccines is exemplified in U.S. Pat. Nos. 5,958,895 and 5,620,896.

The preparation of vaccines that contain polypeptide or peptidesequence(s) as active ingredients is generally well understood in theart, as exemplified by U.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231;4,599,230; 4,596,792; and 4,578,770, all of which are incorporatedherein by reference. Typically, such vaccines are prepared asinjectables either as liquid solutions or suspensions: solid formssuitable for solution in or suspension in liquid prior to injection mayalso be prepared. The preparation may also be emulsified. The activeimmunogenic ingredient is often mixed with excipients that arepharmaceutically acceptable and compatible with the active ingredient.Suitable excipients are, for example, water, saline, dextrose, glycerol,ethanol, or the like and combinations thereof. In addition, if desired,the vaccine may contain amounts of auxiliary substances such as wettingor emulsifying agents, pH buffering agents, or adjuvants that enhancethe effectiveness of the vaccines. In specific embodiments, vaccines areformulated with a combination of substances, as described in U.S. Pat.Nos. 6,793,923 and 6,733,754, which are incorporated herein byreference.

Vaccines may be conventionally administered parenterally, by injection,for example, either subcutaneously or intramuscularly. Additionalformulations which are suitable for other modes of administrationinclude suppositories and, in some cases, oral formulations. Forsuppositories, traditional binders and carriers may include, forexample, polyalkalene glycols or triglycerides: such suppositories maybe formed from mixtures containing the active ingredient in the range ofabout 0.5% to about 10%, preferably about 1% to about 2%. Oralformulations include such normally employed excipients as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate and the like. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders and contain about10% to about 95% of active ingredient, preferably about 25% to about70%.

The polypeptides and polypeptide-encoding DNA constructs may beformulated into a vaccine as neutral or salt forms.Pharmaceutically-acceptable salts include the acid addition salts(formed with the free amino groups of the peptide) and those that areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like.

Typically, vaccines are administered in a manner compatible with thedosage formulation, and in such amount as will be therapeuticallyeffective and immunogenic. The quantity to be administered depends onthe subject to be treated, including the capacity of the individual'simmune system to synthesize antibodies and the degree of protectiondesired. Precise amounts of active ingredient required to beadministered depend on the judgment of the practitioner. However,suitable dosage ranges are of the order of several hundred micrograms ofactive ingredient per vaccination. Suitable regimes for initialadministration and booster shots are also variable, but are typified byan initial administration followed by subsequent inoculations or otheradministrations.

The manner of application may be varied widely. Any of the conventionalmethods for administration of a vaccine are applicable. These arebelieved to include oral application within a solid physiologicallyacceptable base or in a physiologically acceptable dispersion,parenterally, by injection and the like. The dosage of the vaccine willdepend on the route of administration and will vary according to thesize and health of the subject.

In certain instances, it will be desirable to have multipleadministrations of the vaccine, e.g., 2, 3, 4, 5, 6 or moreadministrations. The vaccinations can be at 1, 2, 3, 4, 5, 6, 7, 8, to5, 6, 7, 8, 9, 10, 11, 12 twelve week intervals, including all rangesthere between. Periodic boosters at intervals of 1-5 years will bedesirable to maintain protective levels of the antibodies. The course ofthe immunization may be followed by assays for antibodies against theantigens, as described in U.S. Pat. Nos. 3,791,932; 4,174,384 and3,949,064.

1. Carriers

A given composition may vary in its immunogenicity. It is oftennecessary therefore to boost the host immune system, as may be achievedby coupling a peptide or polypeptide to a carrier. Exemplary andpreferred carriers are keyhole limpet hemocyanin (KLH) and bovine serumalbumin (BSA). Other albumins such as ovalbumin, mouse serum albumin, orrabbit serum albumin can also be used as carriers. Means for conjugatinga polypeptide to a carrier protein are well known in the art and includeglutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester,carbodiimyde, and bis-biazotized benzidine.

2. Adjuvants

The immunogenicity of polypeptide or peptide compositions can beenhanced by the use of non-specific stimulators of the immune response,known as adjuvants. Suitable adjuvants include all acceptableimmunostimulatory compounds, such as cytokines, toxins, or syntheticcompositions. A number of adjuvants can be used to enhance an antibodyresponse against a variant SpA polypeptide or coagulase, or any otherbacterial protein or combination contemplated herein. Adjuvants can (1)trap the antigen in the body to cause a slow release; (2) attract cellsinvolved in the immune response to the site of administration; (3)induce proliferation or activation of immune system cells; or (4)improve the spread of the antigen throughout the subject's body.

Adjuvants include, but are not limited to, oil-in-water emulsions,water-in-oil emulsions, mineral salts, polynucleotides, and naturalsubstances. Specific adjuvants that may be used include IL-1, IL-2,IL-4, IL-7, IL-12, γ-interferon, GMCSP, BCG, aluminum salts, such asaluminum hydroxide or other aluminum compound, MDP compounds, such asthur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A(MPL). RIBI, which contains three components extracted from bacteria,MPL, trehalose dimycolate (TDM), and cell wall skeleton (CWS) in a 2%squalene/Tween 80 emulsion. MHC antigens may even be used. Othersadjuvants or methods are exemplified in U.S. Pat. Nos. 6,814,971,5,084,269, 6,656,462, each of which is incorporated herein byreference).

Various methods of achieving adjuvant affect for the vaccine includesuse of agents such as aluminum hydroxide or phosphate (alum), commonlyused as about 0.05 to about 0.1% solution in phosphate buffered saline,admixture with synthetic polymers of sugars (Carbopol®) used as an about0.25% solution, aggregation of the protein in the vaccine by heattreatment with temperatures ranging between about 70° to about 101° C.for a 30-second to 2-minute period, respectively. Aggregation byreactivating with pepsin-treated (Fab) antibodies to albumin; mixturewith bacterial cells (e.g., C. parvum), endotoxins or lipopolysaccharidecomponents of Gram-negative bacteria; emulsion in physiologicallyacceptable oil vehicles (e.g., mannide mono-oleate (Aracel A)); oremulsion with a 20% solution of a perfluorocarbon (Fluosol-DA®) used asa block substitute may also be employed to produce an adjuvant effect.

Examples of and often preferred adjuvants include complete Freund'sadjuvant (a non-specific stimulator of the immune response containingkilled Mycobacterium tuberculosis), incomplete Freund's adjuvants, andaluminum hydroxide.

In some aspects, it is preferred that the adjuvant be selected to be apreferential inducer of either a Th1 or a Th2 type of response. Highlevels of Th1-type cytokines tend to favor the induction of cellmediated immune responses to a given antigen, while high levels ofTh2-type cytokines tend to favor the induction of humoral immuneresponses to the antigen.

The distinction of Th1 and Th2-type immune response is not absolute. Inreality an individual will support an immune response which is describedas being predominantly Th1 or predominantly Th2. However, it is oftenconvenient to consider the families of cytokines in terms of thatdescribed in murine CD4+ T cell clones by Mosmann and Coffman (Mosmann,and Coffman, 1989). Traditionally, Th1-type responses are associatedwith the production of the INF-γ and IL-2 cytokines by T-lymphocytes.Other cytokines often directly associated with the induction of Th1-typeimmune responses are not produced by T-cells, such as IL-12. Incontrast, Th2-type responses are associated with the secretion of IL-4,IL-5, IL-6, IL-10.

In addition to adjuvants, it may be desirable to co-administer biologicresponse modifiers (BRM) to enhance immune responses. BRMs have beenshown to upregulate T cell immunity or downregulate suppresser cellactivity. Such BRMs include, but are not limited to, Cimetidine (CIM;1200 mg/d) (Smith/Kline, PA); or low-dose Cyclophosphamide (CYP; 300mg/m²) (Johnson/Mead, N.J.) and cytokines such as γ-interferon, IL-2, orIL-12 or genes encoding proteins involved in immune helper functions,such as B-7.

F. Lipid Components and Moieties

In certain embodiments, the present invention concerns compositionscomprising one or more lipids associated with a nucleic acid or apolypeptide/peptide. A lipid is a substance that is insoluble in waterand extractable with an organic solvent. Compounds other than thosespecifically described herein are understood by one of skill in the artas lipids, and are encompassed by the compositions and methods of thepresent invention. A lipid component and a non-lipid may be attached toone another, either covalently or non-covalently.

A lipid may be a naturally occurring lipid or a synthetic lipid.However, a lipid is usually a biological substance. Biological lipidsare well known in the art, and include for example, neutral fats,phospholipids, phosphoglycerides, steroids, terpenes, lysolipids,glycosphingolipids, glucolipids, sulphatides, lipids with ether andester-linked fatty acids and polymerizable lipids, and combinationsthereof.

A nucleic acid molecule or a polypeptide/peptide, associated with alipid may be dispersed in a solution containing a lipid, dissolved witha lipid, emulsified with a lipid, mixed with a lipid, combined with alipid, covalently bonded to a lipid, contained as a suspension in alipid or otherwise associated with a lipid. A lipid orlipid-poxvirus-associated composition of the present invention is notlimited to any particular structure. For example, they may also simplybe interspersed in a solution, possibly forming aggregates which are notuniform in either size or shape. In another example, they may be presentin a bilayer structure, as micelles, or with a “collapsed” structure. Inanother non-limiting example, a lipofectamine(Gibco BRL)-poxvirus orSuperfect (Qiagen)-poxvirus complex is also contemplated.

In certain embodiments, a composition may comprise about 1%, about 2%,about 3%, about 4% about 5%, about 6%, about 7%, about 8%, about 9%,about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%,about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%,about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%,about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%,about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%,about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%,about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, about 99%, or any rangetherebetween, of a particular lipid, lipid type, or non-lipid componentsuch as an adjuvant, antigen, peptide, polypeptide, sugar, nucleic acidor other material disclosed herein or as would be known to one of skillin the art. In a non-limiting example, a composition may comprise about10% to about 20% neutral lipids, and about 33% to about 34% of acerebroside, and about 1% cholesterol. In another non-limiting example,a liposome may comprise about 4% to about 12% terpenes, wherein about 1%of the micelle is specifically lycopene, leaving about 3% to about 11%of the liposome as comprising other terpenes; and about 10% to about 35%phosphatidyl choline, and about 1% of a non-lipid component. Thus, it iscontemplated that compositions of the present invention may comprise anyof the lipids, lipid types or other components in any combination orpercentage range.

G. Combination Therapy

The compositions and related methods of the present invention,particularly administration of a secreted virulence factor or surfaceprotein, including a variant SpA polypeptide or peptide, and/or otherbacterial peptides or proteins to a patient/subject, may also be used incombination with the administration of traditional therapies. Theseinclude, but are not limited to, the administration of antibiotics suchas streptomycin, ciprofloxacin, doxycycline, gentamycin,chloramphenicol, trimethoprim, sulfamethoxazole, ampicillin,tetracycline or various combinations of antibiotics.

In one aspect, it is contemplated that a polypeptide vaccine and/ortherapy is used in conjunction with antibacterial treatment.Alternatively, the therapy may precede or follow the other agenttreatment by intervals ranging from minutes to weeks. In embodimentswhere the other agents and/or a proteins or polynucleotides areadministered separately, one would generally ensure that a significantperiod of time did not expire between the time of each delivery, suchthat the agent and antigenic composition would still be able to exert anadvantageously combined effect on the subject. In such instances, it iscontemplated that one may administer both modalities within about 12-24h of each other or within about 6-12 h of each other. In somesituations, it may be desirable to extend the time period foradministration significantly, where several days (2, 3, 4, 5, 6 or 7) toseveral weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respectiveadministrations.

Various combinations may be employed, for example antibiotic therapy is“A” and the immunogenic molecule given as part of an immune therapyregime, such as an antigen, is “B”:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/BA/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/AA/A/B/A

Administration of the immunogenic compositions of the present inventionto a patient/subject will follow general protocols for theadministration of such compounds, taking into account the toxicity, ifany, of the SpA composition, or other compositions described herein. Itis expected that the treatment cycles would be repeated as necessary. Italso is contemplated that various standard therapies, such as hydration,may be applied in combination with the described therapy.

H. General Pharmaceutical Compositions

In some embodiments, pharmaceutical compositions are administered to asubject. Different aspects of the present invention involveadministering an effective amount of a composition to a subject. In someembodiments of the present invention, staphylococcal antigens, membersof the Ess pathway, including polypeptides or peptides of the Esa or Esxclass, and/or members of sortase substrates may be administered to thepatient to protect against infection by one or more staphylococcuspathogens. Alternatively, an expression vector encoding one or more suchpolypeptides or peptides may be given to a patient as a preventativetreatment. Additionally, such compounds can be administered incombination with an antibiotic or an antibacterial. Such compositionswill generally be dissolved or dispersed in a pharmaceuticallyacceptable carrier or aqueous medium.

In addition to the compounds formulated for parenteral administration,such as those for intravenous or intramuscular injection, otherpharmaceutically acceptable forms include, e.g., tablets or other solidsfor oral administration; time release capsules; and any other formcurrently used, including creams, lotions, mouthwashes, inhalants andthe like.

The active compounds of the present invention can be formulated forparenteral administration, e.g., formulated for injection via theintravenous, intramuscular, sub-cutaneous, or even intraperitonealroutes. The preparation of an aqueous composition that contains acompound or compounds that increase the expression of an MHC class Imolecule will be known to those of skill in the art in light of thepresent disclosure. Typically, such compositions can be prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for use to prepare solutions or suspensions upon the additionof a liquid prior to injection can also be prepared; and, thepreparations can also be emulsified.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil, or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that it may be easily injected. It also should be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms, such as bacteria andfungi.

The proteinaceous compositions may be formulated into a neutral or saltform. Pharmaceutically acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like.

The carrier also can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion, and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques, which yield a powder of the active ingredient, plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Administration of the compositions according to the present inventionwill typically be via any common route. This includes, but is notlimited to oral, nasal, or buccal administration. Alternatively,administration may be by orthotopic, intradermal, subcutaneous,intramuscular, intraperitoneal, intranasal, or intravenous injection. Incertain embodiments, a vaccine composition may be inhaled (e.g., U.S.Pat. No. 6,651,655, which is specifically incorporated by reference).Such compositions would normally be administered as pharmaceuticallyacceptable compositions that include physiologically acceptablecarriers, buffers or other excipients. As used herein, the term“pharmaceutically acceptable” refers to those compounds, materials,compositions, and/or dosage forms which are, within the scope of soundmedical judgment, suitable for contact with the tissues of human beingsand animals without excessive toxicity, irritation, allergic response,or other problem complications commensurate with a reasonablebenefit/risk ratio. The term “pharmaceutically acceptable carrier,”means a pharmaceutically acceptable material, composition or vehicle,such as a liquid or solid filler, diluent, excipient, solvent orencapsulating material, involved in carrying or transporting a chemicalagent.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered, if necessary, and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in isotonic NaCl solution andeither added to hypodermoclysis fluid or injected at the proposed siteof infusion, (see for example, Remington's Pharmaceutical Sciences,1990). Some variation in dosage will necessarily occur depending on thecondition of the subject. The person responsible for administrationwill, in any event, determine the appropriate dose for the individualsubject.

An effective amount of therapeutic or prophylactic composition isdetermined based on the intended goal. The term “unit dose” or “dosage”refers to physically discrete units suitable for use in a subject, eachunit containing a predetermined quantity of the composition calculatedto produce the desired responses discussed above in association with itsadministration, i.e., the appropriate route and regimen. The quantity tobe administered, both according to number of treatments and unit dose,depends on the protection desired.

Precise amounts of the composition also depend on the judgment of thepractitioner and are peculiar to each individual. Factors affecting doseinclude physical and clinical state of the subject, route ofadministration, intended goal of treatment (alleviation of symptomsversus cure), and potency, stability, and toxicity of the particularcomposition.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeutically orprophylactically effective. The formulations are easily administered ina variety of dosage forms, such as the type of injectable solutionsdescribed above.

I. In Vitro, Ex Vivo, or In Vivo Administration

As used herein, the term in vitro administration refers to manipulationsperformed on cells removed from or outside of a subject, including, butnot limited to cells in culture. The term ex vivo administration refersto cells which have been manipulated in vitro, and are subsequentlyadministered to a subject. The term in vivo administration includes allmanipulations performed within a subject.

In certain aspects of the present invention, the compositions may beadministered either in vitro, ex vivo, or in vivo. In certain in vitroembodiments, autologous B-lymphocyte cell lines are incubated with avirus vector of the instant invention for 24 to 48 hours or with avariant SpA and/or cogaulase and/or any other composition describedherein for two hours. The transduced cells can then be used for in vitroanalysis, or alternatively for ex vivo administration. U.S. Pat. Nos.4,690,915 and 5,199,942, both incorporated herein by reference, disclosemethods for ex vivo manipulation of blood mononuclear cells and bonemarrow cells for use in therapeutic applications.

J. Antibodies And Passive Immunization

Another aspect of the invention is a method of preparing animmunoglobulin for use in prevention or treatment of staphylococcalinfection comprising the steps of immunizing a recipient or donor withthe vaccine of the invention and isolating immunoglobulin from therecipient or donor. An immunoglobulin prepared by this method is afurther aspect of the invention. A pharmaceutical composition comprisingthe immunoglobulin of the invention and a pharmaceutically acceptablecarrier is a further aspect of the invention which could be used in themanufacture of a medicament for the treatment or prevention ofstaphylococcal disease. A method for treatment or prevention ofstaphylococcal infection comprising a step of administering to a patientan effective amount of the pharmaceutical preparation of the inventionis a further aspect of the invention.

Inocula for polyclonal antibody production are typically prepared bydispersing the antigenic composition in a physiologically tolerablediluent such as saline or other adjuvants suitable for human use to forman aqueous composition. An immunostimulatory amount of inoculum isadministered to a mammal and the inoculated mammal is then maintainedfor a time sufficient for the antigenic composition to induce protectiveantibodies.

The antibodies can be isolated to the extent desired by well knowntechniques such as affinity chromatography (Harlow and Lane, 1988).Antibodies can include antiserum preparations from a variety of commonlyused animals, e.g. goats, primates, donkeys, swine, horses, guinea pigs,rats or man.

An immunoglobulin produced in accordance with the present invention caninclude whole antibodies, antibody fragments or subfragments. Antibodiescan be whole immunoglobulins of any class (e.g., IgG, IgM, IgA, IgD orIgE), chimeric antibodies or hybrid antibodies with dual specificity totwo or more antigens of the invention. They may also be fragments (e.g.,F(ab′)2, Fab′, Fab, Fv and the like) including hybrid fragments. Animmunoglobulin also includes natural, synthetic, or geneticallyengineered proteins that act like an antibody by binding to specificantigens to form a complex.

A vaccine of the present invention can be administered to a recipientwho then acts as a source of immunoglobulin, produced in response tochallenge from the specific vaccine. A subject thus treated would donateplasma from which hyperimmune globulin would be obtained viaconventional plasma fractionation methodology. The hyperimmune globulinwould be administered to another subject in order to impart resistanceagainst or treat staphylococcal infection. Hyperimmune globulins of theinvention are particularly useful for treatment or prevention ofstaphylococcal disease in infants, immune compromised individuals, orwhere treatment is required and there is no time for the individual toproduce antibodies in response to vaccination.

An additional aspect of the invention is a pharmaceutical compositioncomprising two of more monoclonal antibodies (or fragments thereof;preferably human or humanised) reactive against at least twoconstituents of the immunogenic composition of the invention, whichcould be used to treat or prevent infection by Gram positive bacteria,preferably staphylococci, more preferably S. aureus or S. epidermidis.Such pharmaceutical compositions comprise monoclonal antibodies that canbe whole immunoglobulins of any class, chimeric antibodies, or hybridantibodies with specificity to two or more antigens of the invention.They may also be fragments (e.g., F(ab′)2, Fab′, Fab, Fv and the like)including hybrid fragments.

Methods of making monoclonal antibodies are well known in the art andcan include the fusion of splenocytes with myeloma cells (Kohler andMilstein, 1975; Harlow and Lane, 1988). Alternatively, monoclonal Fvfragments can be obtained by screening a suitable phage display library(Vaughan et al., 1998). Monoclonal antibodies may be humanized or parthumanized by known methods.

EXAMPLES

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion. One skilled in the art will appreciate readilythat the present invention is well adapted to carry out the objects andobtain the ends and advantages mentioned, as well as those objects, endsand advantages inherent herein. The present examples, along with themethods described herein are presently representative of preferredembodiments, are exemplary, and are not intended as limitations on thescope of the invention. Changes therein and other uses which areencompassed within the spirit of the invention as defined by the scopeof the claims will occur to those skilled in the art.

Example 1 Non-Toxigenic Protein a Variants as Subunit Vaccines toPrevent Staphylococcus Aureus Infections

An animal model for S. aureus infection BALB/c mice were infected byintravenous injection with 1×10⁷ CFU of the human clinical isolate S.aureus Newman (Baba et al., 2007). Within 6 hours following infection,99.999% of staphylococci disappeared from the blood stream and weredistributed via the vasculature. Staphylococcal dissemination toperipheral tissues occurred rapidly, as the bacterial load in kidney andother peripheral organ tissues reached 1×10⁵ CFU g⁻¹ within the firstthree hours. The staphylococcal load in kidney tissues increased by 1.5log CFU within twenty-four hours. Forty-eight hours following infection,mice developed disseminated abscesses in multiple organs, detectable bylight microscopy of hematoxylin-eosin stained, thin-sectioned kidneytissue. The initial abscess diameter was 524 μM (±65 μM); lesions wereinitially marked by an influx of polymorphonuclear leukocytes (PMNs) andharbored no discernable organization of staphylococci, most of whichappeared to reside within PMNs. On day 5 of infection, abscessesincreased in size and enclosed a central population of staphylococci,surrounded by a layer of eosinophilic, amorphous material and a largecuff of PMNs. Histopathology revealed massive necrosis of PMNs inproximity to the staphylococcal nidus at the center of abscess lesionsas well as a mantle of healthy phagocytes. A rim of necrotic PMNs wereobserved at the periphery of abscess lesions, bordering eosinophilic,amorphous material that separates healthy renal tissue from lesions.Abscesses eventually reached a diameter of ≧1,524 μM on day 15 or 36. Atlater time intervals, the staphylococcal load was increased to 10⁴-10⁶CFU g⁻¹ and growing abscess lesions migrated towards the organ capsule.Peripheral lesions were prone to rupture, thereby releasing necroticmaterial and staphylococci into the peritoneal cavity or theretroperitoneal space. These events resulted in bacteremia as well as asecondary wave of abscesses, eventually precipitating a lethal outcome.

To enumerate staphylococcal load in renal tissue, animals were killed,their kidneys excised and tissue homogenate spread on agar media forcolony formation. On day 5 of infection, a mean of 1×10⁶ CFU g⁻¹ renaltissue for S. aureus Newman was observed. To quantify abscess formation,kidneys were visually inspected, and each individual organ was given ascore of one or zero. The final sum was divided by the total number ofkidneys to calculate percent surface abscesses (Table 3). In addition,randomly chosen kidneys were fixed in formalin, embedded, thinsectioned, and stained with hematoxylin-eosin. For each kidney, foursagittal sections at 200 μM intervals were viewed by microscopy. Thenumbers of lesions were counted for each section and averaged toquantify the number of abscesses within the kidneys. S. aureus Newmancaused 4.364±0.889 abscesses per kidney, and surface abscesses wereobserved on 14 out of 20 kidneys (70%) (Table 3).

When examined by scanning electron microscopy, S. aureus Newman waslocated in tightly associated lawns at the center of abscesses.Staphylococci were contained by an amorphous pseudocapsule thatseparated bacteria from the cuff of abscesses leukocytes. No immunecells were observed in these central nests of staphylococci, howeveroccasional red blood cells were located among the bacteria. Bacterialpopulations at the abscess center, designated staphylococcal abscesscommunities (SAC), appeared homogenous and coated by an electron-dense,granular material. The kinetics of the appearance of infectious lesionsand the morphological attributes of abscesses formed by S. aureus Newmanwere similar to those observed following mouse infection with S. aureusUSA300 (LAC), the current epidemic community-acquiredmethicillin-resistant S. aureus (CA-MRSA) clone in the United States(Diep et al., 2006).

TABLE 3 Genetic requirements for S. aureus Newman abscess formation inmice Abscess formation in kidney tissue Staphylococcal load in kidneytissue ^(e)Number of ^(a)log₁₀ CFU g⁻¹ ^(b)Significance ^(c)Reduction^(d)Surface abscesses per ^(f) Significance Genotype tissue (P-value)(log₁₀ CFU g⁻¹) abscesses (%) kidney (P-value) wild-type 6.141 ± 0.192 —— 70 4.364 ± 0.889 — ΔsrtA 4.095 ± 0.347 6.7 × 10⁻⁶ 2.046 0 0.000 ±0.000 0.0216 spa 5.137 ± 0.374 0.0144 1.004 13 0.375 ± 0.374 0.0356^(a)Means of staphylococcal load calculated as log₁₀ CFU g⁻¹ inhomogenized renal tissues 5 days following infection in cohorts offifteen BALB/c mice per challenge strain. Standard error of the means(±SEM) is indicated. ^(b)Statistical significance was calculated withthe Students t-test and P-values recorded; P-values < 0.05 were deemedsignificant. ^(c)Reduction in bacterial load calculated as log₁₀ CFUg⁻¹. ^(d)Abscess formation in kidney tissues five days followinginfection was measured by macroscopic inspection (% positive)^(e)Histopathology of hematoxylin-eosin stained, thin sectioned kidneysfrom eight to ten animals; the average number of abscesses per kidneywas recorded and averaged again for the final mean (±SEM).^(f)Statistical significance was calculated with the Students t-test andP-values recorded; P-values < 0.05 were deemed significant.

S. aureus Protein A (spa) mutants are avirulent and cannot formabscesses Sortase A is a transpeptidase that immobilizes nineteensurface proteins in the envelope of S. aureus strain Newman (Mazmanianet al., 1999; Mazmanian et al., 2000). Earlier work identified sortase Aas a virulence factor in multiple animal model systems, however thecontributions of this enzyme and its anchored surface proteins toabscess formation or persistence have not yet been revealed (Jonsson etal., 2002; Weiss et al., 2004). Compared to the wild-type parent (Babaet al., 2007), an isogenic srtA variant (AsrtA) failed to form abscesslesions on either macroscopic or histopathology examination on days 2,5, or 15. In mice infected with the strA mutant, only 1×10⁴ CFU g⁻¹ wasrecovered from kidney tissue on day 5 of infection, which is a 2.046log₁₀ CFU g⁻¹ reduction compared to the wild-type parent strain(P=6.73×10⁻⁶). A similar defect was observed for the srtA mutant of MRSAstrain USA300 (data not shown). Scanning electron microscopy showed thatsrtA mutants were highly dispersed and often associated with leukocytesin otherwise healthy renal tissue. On day fifteen following infection,srtA mutants were cleared from renal tissues, a ≧3.5 log₁₀ CFU g⁻¹reduction compared to the wild-type (Table 3). Thus, sortase A anchoredsurface proteins enable the formation of abscess lesions and thepersistence of bacteria in host tissues, wherein staphylococci replicateas communities embedded in an extracellular matrix and shielded fromsurrounding leukocytes by an amorphous pseudocapsule.

Sortase A anchors a large spectrum of proteins with LPXTG motif sortingsignals to the cell wall envelope, thereby providing for the surfacedisplay of many virulence factors (Mazmanian et al., 2002). To identifysurface proteins required for staphylococcal abscess formation, bursaaurealis insertions were introduced in 5′ coding sequences of genes thatencode polypeptides with LPXTG motif proteins (Bae et al., 2004) andthese mutations were transduced into S. aureus Newman. Mutations in thestructural gene for Protein A (spa) reduced the staphylococcal load ininfected mouse kidney tissues by 1.004 log₁₀ (P=0.0144). When analyzedfor their ability to form abscesses in kidney tissues by histopathology,we observed that the spa mutants were unable to form abscesses ascompared with the wild-type parent strain S. aureus Newman (wild-type S.aureus Newman 4.364±0.889 abscesses per kidney vs. the isogenic spamutant with 0.375±0.374 lesions; P=0.0356).

Protein A blocks innate and adaptive immune responses. Studiesidentified Protein A as a critical virulence factor during thepathogenesis of S. aureus infections. Earlier work demonstrated thatProtein A impedes phagocytosis of staphylococci by binding the Fccomponent of immunoglobulin (Jensen 1958; Uhlén et al., 1984), activatesplatelet aggregation via the von Willebrand factor (Hartleib et al.,2000), functions as a B cell superantigen by capturing the F(ab)₂ regionof VH3 bearing IgM (Roben et al., 1995), and, through its activation ofTNFR1, can initiate staphylococcal pneumonia (Gomez et al., 2004). Dueto the fact that Protein A captures immunoglobulin and displays toxicattributes, the possibility that this surface molecule may function as avaccine in humans has not been rigorously pursued. The inventorsdemonstrate for the first time that Protein A variants no longer able tobind to immunoglobulins, vWF and TNFR-1 are removed of their toxigenicpotential and are able to stimulate humoral immune responses thatprotect against staphylococcal disease.

Molecular basis of Protein A surface display and function. Protein A issynthesized as a precursor in the bacterial cytoplasm and secreted viaits YSIRK signal peptide at the cross wall, i.e., the cell divisionseptum of staphylococci (FIG. 1A). (DeDent et al., 2007; DeDent et al.,2008). Following cleavage of the C-terminal LPXTG sorting signal,Protein A is anchored to bacterial peptidoglycan crossbridges by sortaseA (Schneewind et al., 1995; Mazmanian et al., 1999; Mazmanian et al.,2000). Protein A is the most abundant surface protein of staphylococci;the molecule is expressed by virtually all S. aureus strains (Said-Salimet al., 2003; Cespedes et al., 2005; Kennedy et al., 2008).Staphylococci turn over 15-20% of their cell wall per division cycle(Navarre and Schneewind 1999). Murine hydrolases cleave the glycanstrands and wall peptides of peptidoglycan, thereby releasing Protein Awith its attached C-terminal cell wall disaccharide tetrapeptide intothe extracellular medium (Ton-That et al., 1999). Thus, by physiologicaldesign, Protein A is both anchored to the cell wall and displayed on thebacterial surface but also released into surrounding tissues during hostinfection (Marraffini et al., 2006).

Protein A captures immunoglobulins on the bacterial surface and thisbiochemical activity enables staphylococcal escape from host innate andacquired immune responses (Jensen 1958; Goodyear and Silverman 2004).Interestingly, region X of Protein A (Guss et al., 1984), a repeatdomain that tethers the IgG binding domains to the LPXTG sortingsignal/cell wall anchor, is perhaps the most variable portion of thestaphylococcal genome (Schneewind et al., 1992; Said-Salim et al.,2003). Each of the five immunoglobulin binding domains of Protein A(SpA), formed from three helix bundles and designated E, D, A, B, and C,exerts similar structural and functional properties (Sjödahl 1977;Jansson et al., 1998). The solution and crystal structure of domain Dhas been solved both with and without the Fc and V_(H)3 (Fab) ligands,which bind Protein A in a non-competitive manner at distinct sites(Graille et al., 2000).

In the crystal structure complex, the Fab interacts with helix II andhelix III of domain D via a surface composed of four VH region β-strands(Graille et al., 2000). The major axis of helix II of domain D isapproximately 50° to the orientation of the strands, and theinterhelical portion of domain D is most proximal to the C0 strand. Thesite of interaction on Fab is remote from the Ig light chain and theheavy chain constant region. The interaction involves the followingdomain D residues: Asp-36 of helix II as well as Asp-37 and Gln-40 inthe loop between helix II and helix III, in addition to several otherresidues with SpA-D (Graille et al., 2000). Both interacting surfacesare composed predominantly of polar side chains, with three negativelycharged residues on domain D and two positively charged residues on the2A2 Fab buried by the interaction, providing an overall electrostaticattraction between the two molecules. Of the five polar interactionsidentified between Fab and domain D, three are between side chains. Asalt bridge is formed between Arg-H19 and Asp-36 and two hydrogen bondsare made between Tyr-H59 and Asp-37 and between Asn-H82a and Ser-33.Because of the conservation of Asp-36 and Asp-37 in all five IgG bindingdomains of Protein A, these residues were selected for mutagenesis.

The SpA-D sites responsible for Fab binding are structurally separatefrom the domain surface that mediates Fcγ binding. The interaction ofFcγ with domain B primarily involves residues in helix I with lesserinvolvement of helix II (Deisenhofer 1981; Gouda et al., 1992). With theexception of the Gln-32, a minor contact in both complexes, none of theresidues that mediate the Fcγ interaction are involved in Fab binding.To examine the spatial relationship between these different Ig-bindingsites, the SpA domains in these complexes have been superimposed toconstruct a model of a complex between Fab, the SpA-domain D, and theFcγ molecule. In this ternary model, Fab and Fcγ form a sandwich aboutopposite faces of the helix II without evidence of steric hindrance ofeither interaction. These findings illustrate how, despite its smallsize (i.e., 56-61 aa), a SpA domain can simultaneously display bothactivities, explaining experimental evidence that the interactions ofFab with an individual domain are noncompetitive. Residues for theinteraction between SpA-D and Fcγ are Gln-9 and Gln-10.

In contrast, occupancy of the Fc portion of IgG on the domain D blocksits interaction with vWF A1 and probably also TNFR1 (O'Seaghdha et al.,2006). Mutations in residues essential for IgG Fc binding (F5, Q9, Q10,S11, F13, Y14, L17, N28, I31 and K35) are also required for vWF A1 andTNFR1 binding (Cedergren et al., 1993; Gomez et al., 2006; O'Seaghdha etal. 2006), whereas residues critical for the V_(H)3 interaction (Q26,G29, F30, S33, D36, D37, Q40, N43, E47) have no impact on the bindingactivities of IgG Fc, vWF A1 or TNFR1 (Jansson et al., 1998; Graille etal., 2000). The Protein A immunoglobulin Fab binding activity targets asubset of B cells that express VH3 family related IgM on their surface,i.e. these molecules function as VH3 type B cell receptors (Roben etal., 1995). Upon interaction with SpA, these B cells rapidly proliferateand then commit to apoptosis, leading to preferential and prolongeddeletion of innate-like B lymphocytes (i.e. marginal zone B cells andfollicular B2 cells) (Goodyear and Silverman 2003; Goodyear andSilverman 2004). It is important to note that more than 40% ofcirculating B cells are targeted by the Protein A interaction and theVH3 family represents the largest family of human B cell receptors toimpart protective humoral responses against pathogens (Goodyear andSilverman 2003; Goodyear and Silverman 2004). Thus, Protein A functionsanalogously to staphylococcal superantigens (Roben et al., 1995), albeitthat the latter class of molecules, for example SEB, TSST-1, TSST-2,form complexes with the T cell receptor to inappropriately stimulatehost immune responses and thereby precipitating characteristic diseasefeatures of staphylococcal infections (Roben et al., 1995; Tiedemann etal., 1995). Together these findings document the contributions ofProtein A in establishing staphylococcal infections and in modulatinghost immune responses.

Non-toxigenic variant of Protein A. The inventors have developed anon-toxigenic variant of staphylococcal Protein A and, with this reagentin hand, aimed for the first time to measure the immune response ofanimals to Protein A immunization. Further, the inventors addresswhether immunization of animals with a non-toxigenic variant of ProteinA could generate immune responses that raise protective immunity againststaphylococcal infection.

To perturb the IgG Fc, vWF A1 and TNFR1 binding activities of Protein A,glutamine (Q) residues 9 and 10 [the numbering here is derived from thatestablished for the SpA domain D] were modified generating lysine orglycine substitutions for both glutamines with the expectation thatthese substitutions abolish the ion bonds formed between wild-typeProtein A and its ligands. The added effect of the dual lysinesubstitutions may be that these positively charged residues institute arepellent charge for immunoglobulins. To perturb IgM Fab VH3 binding,the inventors selected the aspartate (D) residues 36 and 37 of SpA-D,each of which is required for the association of Protein A with the Bcell receptor. D36 and D37 were both substituted with alanine The Q9,10Kand D36,37A mutations were combined in the recombinant moleculeSpA-D_(Q9,10K;D36,37A) and examined for the binding attributes ofProtein A.

In brief, the Protein A (spa) genomic sequence of Staphylococcus aureusN315 was PCR amplified with the primers(GCTGCACATATGGCGCAACACGATGAAGCTCAAC [5′ primer](SEQ ID NO:35) andAGTGGATCCTTATGCTTTGTTAGCATCTGC [3′ primer] (SEQ ID NO:36)), cloned intothe pET15b vector (pYSJ1, codons 48-486) (Stranger-Jones, et al., 2006)and recombinant plasmid transformed into E. coli BL21(DE3) (Studier etal., 1990). The Protein A product derived from pYSJ1 harbors SpAresidues 36-265 fused to the N-terminal His tag (MGSSHHHHHHSSGLVPRGS(SEQ ID NO:37)). Following IPTG inducible expression, recombinantN-terminal His₆-tagged SpA was purified by affinity chromatography onNi-NTA resin (Stranger-Jones et al., 2006). The domain D of SpA (SpA-D)was PCR amplified with a pair of specific primers(AACATATGTTCAACAAAGATCAACAAAGC [5′ primer](SEQ ID NO:38) andAAGGATCCAGATTCGTTTAATTTTTTAGC [3′ primer] (SEQ ID NO:39)), sub-clonedinto the pET15b vector (pHAN1, spa codons 212-261) and recombinantplasmid transformed into E. coli BL21(DE3) to express and purifyrecombinant N-terminal His₆-tagged protein. To generate mutations in theSpA-D coding sequence, sets of two pairs of primers were synthesized(for D to A substitutions: CTTCATTCAAAGTCTTAAAGCCGCCCCAAGCCAAAGCACTAAC[5′ primer] (SEQ ID NO:40) andGTTAGTGCTTTGGCTTGGGGCGGCTTTAAGACTTTGAATGAAG [3′ primer] (SEQ ID NO:41);for Q to K substitutions CATATGTTCAACAAAGATAAAAAAAGCGCCTTCTATGAAATC [5′primer] (SEQ ID NO:42) and GATTTCATAGAAGGCGCTTTTTTTATCTTTGTTGAACATATG[3′ primer] (SEQ ID NO:43); for Q to G substitutionsCATATGTTCAACAAAGATGGAGGAAGCGCCTTCTATGAAATC [5′ primer] (SEQ ID NO:44)and GATTTCATAGAAGGCGCTTCCTCCATCTTTGTTGAACATAT G′ [3′ primer] (SEQ IDNO:45). Primers were used for quick-change mutagenesis protocols.Following mutagenesis, DNA sequences were confirmed for each of therecombinant proteins: SpA, SpA-D and SpA-D_(Q9,10G;D36,37A) andSpA-D_(Q9,10K;D36,37A). All proteins were purified from lysates ofrecombinant E. coli using Ni-NTA chromatography and subsequentlydialyzed against PBS and stored at 4° C.

To measure binding of immunoglobulin to Protein A and its variants, 200μg of purified protein was diluted into a 1 ml volume using columnbuffer (50 mM Tris-HCl, 150 mM NaCl, pH7.5) and then loaded onto apre-equilibrated Ni-NTA column (1 ml bed volume). Columns were washedwith 10 ml of column buffer. 200 μg of purified human IgG was diluted ina total volume of 1 ml column buffer and then applied to each of thecolumns charged with Protein A and its variants. The columns weresubsequently washed with 5 ml wash buffer (10 mM imidazole in columnbuffer) and 5 ml column buffer. Protein samples were eluted with 2 mlelution buffer (500 mM imidazole in column buffer), fractions collectedand aliquots subjected to SDS-PAGE gel electrophoresis, followed byCoomassie-Blue staining As shown in FIG. 1C, wild-type Protein A (SpA)and its SpA-domain D both retained immunogobulin during chromatography.In contrast, the SpA-D_(DQ9,10K;D36,37A) variant did not bind toimmunoglobulin.

To quantify the binding of Protein A and its variants to the Fc portionof immunoglobulin and the VH3 domain of Fab, HRP conjugated humanimmunoglobulin G [hIgG], the Fc portion of human IgG [hFc] and theF(ab)₂ portion of human IgG [hF(ab)₂] as well as ELISA assays were usedto quantify the relative amount binding to Protein A and its variants.The data in FIG. 1D demonstrate the binding of SpA and SpA-D to hIgG andhFc, whereas SpA-D_(Q9,10G;D36,37A) and SpA-D_(Q9,10K;D36,37A) displayedonly background binding activities. SpA bound similar amounts of hFc andhF(ab)₂, however the binding of SpA-D to hF(ab)₂ was reduced compared tofull length SpA. This result suggests that the presence of multiple IgGbinding domains may cooperatively increase the ability of Protein A tobind to the B cell receptor. When compared with the reduced bindingpower of SpA-D for hF(ab)₂, of the two variants onlySpA-D_(Q9,10K;D36,37A) displayed a significant reduction in the abilityto bind the VH3 domain of immunoglobulin. To examine the toxigenicattributes of SpA-D and its variants, purified proteins were injectedinto mice, which were sacrificed after 4 hours to remove their spleens.Organ tissue was homogenized, capsular material removed and B cellsstained with fluorescent CD19 antibodies. Following FACS analysis toquantify the abundance of B cells in splenic tissues, it was observedthat SpA-D caused a 5% drop in the B cell count compared to a mock (PBS)control (FIG. 1E). In contrast, SpA-D_(Q9,10K;D36,37A) did not cause areduction in B-cell counts, indicating that the mutant molecule had lostits toxigenic attributes of stimulating B cell proliferation and death(FIG. 1E). In summary, amino acid substitutions in the SpA-D residuesQ9, Q10, D36, and D37 abolished the ability of Protein A domains to bindimmunoglobulins or exert toxigenic functions in human and animaltissues.

Non-toxigenic Protein A variants elicit vaccine protection. To testwhether or not Protein A and its variants can function as vaccineantigens, SpA, SpA-D, SpA-D_(Q9,10K;D36,37A), and SpA-D_(Q9,10K;D36,37A)were emulsified with complete or incomplete Freund's adjuvant andimmunized 4 week old BALB/c mice on day 1 and day 11 with 50 μg ofpurified protein. Cohort of animals (n=5) were analyzed for humoralimmune responses to immunization by bleeding the animals before (day 0)and after the immunization schedule (day 21). Table 4 indicates thatimmunized mice generated only a modest humoral immune response directedat wild-type Protein A or its SpA-D module, whereas the amount ofantibody raised following immunization with SpA-D_(Q9,10K;D36,37A) orSpA-D_(Q9,10K;D36,37A) was increased four to five fold. Followingintravenous challenge with 1×10⁷ CFU S. aureus Newman, animals werekilled on day 4, their kidneys removed and either analyzed forstaphylococcal load (by plating tissue homogenate on agar plates andenumerating colony forming units, CFU) or histopathology. As expected,mock (PBS) immunized mice (n=19) harbored 6.46 log₁₀ (±0.25) CFU inkidney tissue and infectious lesions were organized into 3.7 (±1.2)abscesses per organ (n=10) (Table 4). Immunization of animals with SpAled to a 2.51 log₁₀ CFU reduction on day 5 (P=0.0003) with 2.1 (±1.2)abscesses per organ. The latter data indicate that there was nosignificant reduction in abscess formation (P=0.35). Immunization withSpA-D generated similar results: a 2.03 log₁₀ CFU reduction on day 5(P=0.0001) with 1.5 (±0.8) abscesses per organ (P=0.15). In contrast,immunization with SpA-D_(Q9,10K;D36,37A) or SpA-D_(Q9,10G;D36,37A)created increased protection, with 3.07 log₁₀ and 3.03 log₁₀ CFUreduction on day 4, respectively (statistical significance P<0.0001 forboth observations). Further, immunization with bothSpA-D_(Q9,10K;D36,37A) and SpA-D_(Q9,10G;D36,37A) generated significantprotection from staphylococcal abscess formation, as only 0.5 (±0.4) and0.8 (±0.5) infectious lesions per organ (P=0.02 and P=0.04) wereidentified. Thus, immunization with non-toxigenic Protein A variantsgenerates increased humoral immune responses for Protein A and providesprotective immunity against staphylococcal challenge. These dataindicate that Protein A is an ideal candidate for a human vaccine thatprevents S. aureus disease.

These exciting results have several implications for the design of ahuman vaccine. First, the generation of substitution mutations thataffect the ability of the immunoglobulin binding domains of Protein A,either alone or in combination of two or more domains, can generatenon-toxigenic variants suitable for vaccine development. It seems likelythat a combination of mutant IgG binding domains closely resembling thestructure of Protein A can generate even better humoral immune responsesas is reported here for the SpA-domain D alone. Further, a likelyattribute of Protein A specific antibodies may be that the interactionof antigen binding sites with the microbial surface can neutralize theability of staphylococci to capture immunoglobulins via their Fc portionor to stimulate the B cell receptor via the VH3 binding activities.

TABLE 4 Non-toxigenic Protein A variants as vaccine antigens thatprevent S. aureus disease Bacterial load in kidney (n = number of mice)Abscess formation in mice (n = number of mice) ^(a)log₁₀ CFU IgG^(d)Surface ^(f)P Antigen g⁻¹ ^(b)Reduction ^(c)P value titer abscessReduction ^(e)Histopathology Reduction value Mock 6.46 ± 0.25 — — <10014/19 — 3.7 ± 1.2 — — (n = 19) (70%) (n = 10) SpA 3.95 ± 0.56 2.510.0003 1706 ± 370 10/20 32% 2.1 ± 1.2 2.2 0.35 (n = 20) (50%) (n = 10)SpA-D 4.43 ± 0.41 2.03 0.0001 381 ± 27 10/18 25% 1.5 ± 0.8 2.2 0.15 (n =18) (55%) (n = 10) SpA-D1 3.39 ± 0.50 3.07 <0.0001 5600 ± 801  6/20 59%0.5 ± 0.4 3.2 0.02 (n = 19) (30%) (n = 10) SpA-D2 3.43 ± 0.46 3.03<0.0001 3980 ± 676  6/19 57% 0.8 ± 0.5 2.9 0.04 (n = 19) (32%) (n = 10)^(a)Means of staphylococcal load calculated as log₁₀ CFU g⁻¹ inhomogenized renal tissues 4 days following infection in cohorts of 18 to20 BALB/c mice. Standard error of the means (±SEM) is indicated.^(c)Statistical significance was calculated with the Students t-test andP-values recorded; P-values < 0.05 were deemed significant.^(b)Reduction in bacterial load calculated as log₁₀ CFU g⁻¹. ^(d)Abscessformation in kidney tissues four days following infection was measuredby macroscopic inspection (% positive) ^(e)Histopathology ofhematoxylin-eosin stained, thin sectioned kidneys from ten animals; thenumber of abscesses per kidney was recorded and averaged for the finalmean (±SEM). ^(f)Statistical significance was calculated with theStudents t-test and P-values recorded; P-values < 0.05 were deemedsignificant. SpA-D1 and SpA-D2 represent SpA-D_(Q9, 10K; D36, 37A) andSpA-D_(Q9, 10G; D36, 37A), respectively.

Vaccine protection in murine abscess, murine lethal infection, andmurine pneumonia models. Three animal models have been established forthe study of S. aureus infectious disease. These models are used here toexamine the level of protective immunity provided via the generation ofProtein A specific antibodies.

Materials and Methods

Murine abscess—BALB/c mice (24-day-old female, 8-10 mice per group,Charles River Laboratories, Wilmington, Mass.) are immunized byintramuscular injection into the hind leg with purified protein (Changet al., 2003; Schneewind et al., 1992). Purified SpA, SpA-D orSpA-DQ9,10K;D36,37A (50 μg protein) is administered on days 0(emulsified 1:1 with complete Freund's adjuvant) and 11 (emulsified 1:1with incomplete Freund's adjuvant). Blood samples are drawn byretroorbital bleeding on days 0, 11, and 20. Sera are examined by ELISAfor IgG titers for specific SpA-D and SpA-DQ9,10K;D36,37A bindingactivity. Immunized animals are challenged on day 21 by retroorbitalinjection of 100 μl of S. aureus Newman or S. aureus USA300 suspension(1×10⁷ cfu). For this, overnight cultures of S. aureus Newman arediluted 1:100 into fresh tryptic soy broth and grown for 3 h at 37° C.Staphylococci are centrifuged, washed twice, and diluted in PBS to yieldan A₆₀₀ of 0.4 (1×10⁸ cfu per ml). Dilutions are verified experimentallyby agar plating and colony formation. Mice are anesthetized byintraperitoneal injection of 80-120 mg of ketamine and 3-6 mg ofxylazine per kilogram of body weight and infected by retroorbitalinjection. On day 5 or 15 following challenge, mice are euthanized bycompressed CO₂ inhalation. Kidneys are removed and homogenized in 1%Triton X-100. Aliquots are diluted and plated on agar medium fortriplicate determination of cfu. For histology, kidney tissue isincubated at room temperature in 10% formalin for 24 h. Tissues areembedded in paraffin, thin-sectioned, stained with hematoxylinleosin,and examined by microscopy.

Murine lethal infection—BALB/c mice (24-day-old female, 8-10 mice pergroup, Charles River Laboratories, Wilmington, Mass.) are immunized byintramuscular injection into the hind leg with purified SpA, SpA-D orSpA-D_(Q9,10K;D36, 37A) (50 μg protein). Vaccine is administered on days0 (emulsified 1:1 with complete Freund's adjuvant) and 11 (emulsified1:1 with incomplete Freund's adjuvant). Blood samples are drawn byretroorbital bleeding on days 0, 11, and 20. Sera are examined by ELISAfor IgG titers with specific SpA-D and SpA-D_(Q9,10K;D36,37A) bindingactivity. Immunized animals are challenged on day 21 by retroorbitalinjection of 100 μl of S. aureus Newman or S. aureus USA300 suspension(15×10⁷ cfu) (34). For this, overnight cultures of S. aureus Newman arediluted 1:100 into fresh tryptic soy broth and grown for 3 h at 37° C.Staphylococci are centrifuged, washed twice, diluted in PBS to yield anA₆₀₀ of 0.4 (1×10⁸ cfu per ml) and concentrated. Dilutions are verifiedexperimentally by agar plating and colony formation. Mice areanesthetized by intraperitoneal injection of 80-120 mg of ketamine and3-6 mg of xylazine per kilogram of body weight. Immunized animals arechallenged on day 21 by intraperitoneal inject with 2×10¹⁰ cfu of S.aureus Newman or 3−10×10⁹ cfu of clinical S. aureus isolates. Animalsare monitored for 14 days, and lethal disease is recorded.

Murine pneumonia model—S. aureus strains Newman or USA300 (LAC) aregrown at 37° C. in tryptic soy broth/agar to OD₆₆₀ 0.5. 50-ml culturealiquots are centrifuged, washed in PBS, and suspended in 750 μl PBS formortality studies (3-4×10⁸ CFU per 30-μl volume), or 1,250 μl PBS (2×10⁸CFU per 30-μl volume) for bacterial load and histopathology experiments(2, 3). For lung infection, 7-wk-old C57BL/6J mice (The JacksonLaboratory) are anesthetized before inoculation of 30 μl of S. aureussuspension into the left nare. Animals are placed into the cage in asupine position for recovery and observed for 14 days. For activeimmunization, 4-wk-old mice receive 20 μg SpA-D orSpA-D_(Q9,10K;D36,37A) in CFA on day 0 via the i.m. route, followed by aboost with 20 μg SpA-D or SpA-D_(Q9,10K;D36,37A) in incomplete Freund'sadjuvant (IFA) on day 10. Animals are challenged with S. aureus on day21. Sera are collected before immunization and on day 20 to assessspecific antibody production. For passive immunization studies, 7-wk-oldmice receive 100 μl of either NRS (normal rabbit serum) orSpA-D-specific rabbit antisera via i.p. injection 24 h before challenge.To assess the pathological correlates of pneumonia, infected animals arekilled via forced CO₂ inhalation before removal of both lungs. The rightlung is homogenized for enumeration of lung bacterial load. The leftlung is placed in 1% formalin and paraffin embedded, thin sectioned,stained with hematoxylin-eosin, and analyzed by microscopy.

Rabbit antibodies—Purified 200 μg SpA-D or SpA-D_(Q9,10K;D36,37A) isused as an immunogen for the production of rabbit antisera. 200 μgprotein is emulsified with CFA for injection at day 0, followed bybooster injections with 200 μg protein emulsified with IFA on days 21and 42. Rabbit antibody titers are determined by ELISA. Purifiedantibodies are obtained by affinity chromatography of rabbit serum onSpA-D or SpA-D_(Q9,10K;D36,37A) sepharose. The concentration of elutedantibodies is measured by absorbance at A₂₈₀ and specific antibodytiters are determined by ELISA.

Active immunization with SpA-domain D variants.—To determine vaccineefficacy, animals are actively immunized with purified SpA-D orSpAD_(Q9,10K;D36,37A). As a control, animals are immunized with adjuvantalone. Antibody titers against Protein A preparations are determinedusing SpA-D or SpA-D_(Q9,10K;D36,37A) as antigens; note that theSpA-D_(Q9,10K;D36,37A) variant cannot bind the Fc or Fab portion of IgG.Using infectious disease models described above, any reduction inbacterial load (murine abscess and pneumonia), histopathology evidenceof staphylococcal disease (murine abscess and pneumonia) and protectionfrom lethal disease (murine lethal challenge and pneumonia) is measured.

Passive immunization with affinity purified rabbit polyclonal antibodiesgenerated against SpA-domain D variants. To determine protectiveimmunity of Protein A specific rabbit antibodies, mice are passivelyimmunized with 5 mg/kg of purified SpA-D or SpA-D_(Q9,10K;D36,37A)derived rabbit antibodies. Both of these antibody preparations arepurified by affinity chromatography using immobilized SpA-D orSpA-D_(Q9,10K;D36,37A). As a control, animals are passively immunizedwith rV10 antibodies (a plague protective antigen that has no impact onthe outcome of staphylococcal infections). Antibody titers against allProtein A preparations are determined using SpA-D_(Q9,10K;D36,37A) as anantigen, as this variant cannot bind the Fc or Fab portion of IgG. Usingthe infectious disease models described above, the reduction inbacterial load (murine abscess and pneumonia), histopathology evidenceof staphylococcal disease (murine abscess and pneumonia), and theprotection from lethal disease (murine lethal challenge and pneumonia)is measured.

Example 2 Non-Toxigenic Protein a Vaccine for Methicillin-ResistantStaphylococcus Aureus Infection

Clinical isolates of S. aureus express protein A (Shopsin et al., 1999,whose primary translational product is comprised of an N-terminal signalpeptide (DeDent et al., 2008), five Ig-BDs (designated E, D, A, B and C)(Sjodahl, 1977), region X with variable repeats of an eight residuepeptide (Guss et al., 1984), and C-terminal sorting signal for the cellwall anchoring of SpA (Schneewind et al., 1992; Schneewind et al., 1995)(FIG. 1A-1B). Guided by amino acid homology (Uhlen et al., 1984), thetriple α-helical bundle structure of IgBDs (Deisenhofer et al., 1978;Deisenhofer et al., 1981) and their atomic interactions with Fab V_(H)3(Graille et al., 2000) or Fcγ (Gouda et al., 1998), glutamine 9 and 10were selected as well as aspartate 36 and 37 as critical for theassociation of SpA with antibodies or B cell receptor, respectively.Substitutions Gln9Lys, Gln10Lys, Asp36Ala and Asp37Ala were introducedinto the D domain to generate SpA-D_(KKAA) (FIG. 1B). The ability ofisolated SpA-D or SpA-D_(KKAA) to bind human IgG was analyzed byaffinity chromatography (FIG. 1D). Polyhistidine tagged SpA-D as well asfull-length SpA retained human IgG on Ni-NTA, whereas SpA-D_(KKAA) and anegative control (SrtA) did not (FIG. 1C). A similar result was observedwith von Willebrand factor (Hartleib et al., 2000), which, along withtumor necrosis factor receptor 1 (TNFR1) (Gomez et al., 2004), can alsobind protein A via glutamine 9 and 10 (FIG. 1D). Human immunoglobulinencompasses 60-70% V_(H)3-type IgG. The inventors distinguish between Fcdomain and B cell receptor activation of Igs and measured association ofhuman Fcγ and F(ab)₂ fragments, both of which bound to full-length SpAor SpA-D, but not to SpA-D_(KKAA) (FIG. 1D). Injection of SpA-D into theperitoneal cavity of mice resulted in B cell expansion followed byapoptotic collapse of CD19+ lymphocytes in spleen tissue of BALB/c mice(Goodyear and Silverman, 2003)(FIG. 1E). B cell superantigen activitywas not observed following injection with SpA-D_(KKAA), andTUNEL-staining of splenic tissue failed to detect the increase inapoptotic cells that follows injection of SpA or SpA-D (FIG. 1E).

Antibodies against SpA-D_(AA) protect against MSSA and MRSA infections.Naive six week old BALB/c mice were injected with 50 μg each of purifiedSpA, SpA-D or SpA-D_(KKAA) emulsified in CFA and boosted with the sameantigen emulsified in IFA. In agreement with the hypothesis that SpA-Dpromotes the apoptotic collapse of activated clonal B cell populations,the inventors observed a ten-fold higher titer of SpA-D_(KKAA) specificantibodies following immunization of mice with the non-toxigenic variantas compared to the B cell superantigen (Spa-D vs. SpA-D_(KKAA) P<0.0001,Table 5). Antibody titers raised by immunization with full-length SpAwere higher than those elicited by SpA-D (P=0.0022), which is likely dueto the larger size and reiterative domain structure of this antigen(Table 5). Nevertheless, even SpA elicited lower antibody titers thanSpA-D_(KKAA) (P=0.0003), which encompasses only 50 amino acids ofprotein A (520 residues, SEQ ID NO:33). Immunized mice were challengedby intravenous inoculation with S. aureus Newman and the ability ofstaphylococci to seed abscesses in renal tissues was examined bynecropsy four days after challenge. In homogenized renal tissue of mock(PBS/adjuvant) immunized mice, an average staphylococcal load of 6.46log₁₀ CFU g⁻¹ was enumerated (Table 5). Immunization of mice with SpA orSpA-D led to a reduction in staphylococcal load, however SpA-D_(KKAA)vaccinated animals displayed an even greater, 3.07 log₁₀ CFU g⁻¹reduction of S. aureus Newman in renal tissues (P <0.0001, Table 5).Abscess formation in kidneys was analyzed by histopathology (FIG. 2).Mock immunized animals harbored an average of 3.7 (±1.2) abscesses perkidney (Table 5). Vaccination with SpA-D_(KKAA) reduced the averagenumber of abscesses to 0.5 (±0.4) (P=0.0204), whereas immunization withSpA or SpA-D did not cause a significant reduction in the number ofabscess lesions (Table 5). Lesions from SpA-D_(KKAA) vaccinated animalswere smaller in size, with fewer infiltrating PMNs andcharacteristically lacked staphylococcal abscess communities (Cheng etal., 2009) (FIG. 2). Abscesses in animals that had been immunized withSpA or SpA-D displayed the same overall structure of lesions in mockimmunized animals (FIG. 2).

The inventors examined whether SpA-D_(KKAA) immunization can protectmice against MRSA strains and selected the USA300 LAC isolate for animalchallenge (Diep et al., 2006). This highly virulent CA-MRSA strainspread rapidly throughout the United States, causing significant humanmorbidity and mortality (Kennedy et al., 2008). Compared to adjuvantcontrol mice, SpA-D_(KKAA) immunized animals harbored a 1.07 log₁₀ CFUg⁻¹ reduction in bacterial load of infected kidney tissues.Histopathology examination of renal tissue following S. aureus USA300challenge revealed that the average number of abscesses was reduced from4.04 (±0.8) to 1.6 (±0.6) (P=0.02774). In contrast, SpA or SpA-Dimmunization did not cause a significant reduction in bacterial load orabscess formation (Table 5).

SpA-D_(KKAA) antibodies prevent immunoglobulin-protein A interaction.Rabbits were immunized with SpA-D_(KKAA) and specific antibodies werepurified on SpA-D_(KKAA) affinity column followed by SDS-PAGE (FIG. 3).SpA-D_(KKAA) specific IgG was cleaved with pepsin to generate Fcγ andF(ab)₂ fragments, the latter of which were purified by chromatography onSpA-D_(KKAA) column (FIG. 3). Binding of human IgG or vWF to SpA orSpA-D was perturbed by SpA-D_(KKAA) specific F(ab)₂, indicating thatSpA-D_(KKAA) derived antibodies neutralize the B cell superantigenfunction of protein A as well as its interactions with Ig (FIG. 3).

SpA_(KKAA) generates improved protective immune responses. To furtherimprove the vaccine properties for non-toxigenic protein A, theinventors generated SpA_(KKAA), which includes all five IgBDs with fouramino acid substitutions—substitutions corresponding to Gln9Lys,Gln10Lys, Asp36Ala and Asp37Ala of domain D—in each of its five domains(E, D, A, B and C). Polyhistidine tagged SpA_(KKAA) was purified byaffinity chromatography and analyzed by Coomassie Blue-stained SDS-PAGE(FIG. 4). Unlike full-length SpA, SpA_(KKAA) did not bind human IgG, Fcand F(ab)₂ or vWF (FIG. 4). SpA_(KKAA) failed to display B cellsuperantigen activity, as injection of the variant into BALB/c mice didnot cause a depletion of CD19+ B cells in splenic tissue (FIG. 4).SpA_(KKAA) vaccination generated higher specific antibody titers thanSpA-D_(KKAA) immunization and provided mice with elevated protectionagainst S. aureus USA300 challenge (Table 5). Four days followingchallenge, SpA_(KKAA) vaccinated animals harbored 3.54 log₁₀ CFU g⁻¹fewer staphylococci in renal tissues (P=0.0001) and also caused agreater reduction in the number of abscess lesions (P=0.0109) (Table 5).As a test whether protein A vaccines impact other MRSA strains, micewere challenged with the Japanese vancomycin-resistant MRSA isolate Mu50(Hiramatsu et al., 1997). Similar to the data observed with the MRSAisolate USA300, SpA_(KKAA) vaccinated animals harbored fewer Mu50staphylococci in renal tissues than mock immunized animals (P=0.0248,FIG. 7).

Passive transfer of SpA-specific antibodies prevents staphylococcaldisease. SpA_(KKAA) was used to immunize rabbits. Rabbit antibodiesspecific for SpA-D_(KKAA) or SpA_(KKAA) were affinity purified onmatrices with immobilized cognate antigen and injected at aconcentration of 5 mg kg⁻¹ body weight into the peritoneal cavity ofBALB/c mice (Table 6). Twenty-four hours later, specific antibody titerswere determined in serum and animals challenged by intravenousinoculation with S. aureus Newman. Passive transfer reduced thestaphylococcal load in kidney tissues for SpA-D_(KKAA) (P=0.0016) orSpA_(KKAA) (P=0.0005) specific antibodies. On histopathologyexamination, both antibodies reduced the abundance of abscess lesions inthe kidneys of mice challenged with S. aureus Newman (Table 6). Togetherthese data reveal that vaccine protection following immunization withSpA-D_(KKAA) or SpA_(KKAA) is conferred by antibodies that neutralizeprotein A.

The inventors also sought to ascertain whether protein A-specificantibodies can protect animals against lethal challenge. BALB/c micewere actively or passively immunized to raise antibodies againstSpA_(KKAA) and then challenged by intraperitoneal injection with lethaldoses of S. aureus Newman (FIG. 6). Antibodies against SpA_(KKAA),whether raised by active (P=0.0475, SpA_(KKAA) vs. mock) or passiveimmunization (P=0.0493, SpA_(KKAA) vs. mock), conferred protectionagainst lethal challenge with S. aureus Newman (FIG. 6)

TABLE 5 Active immunization of mice with protein A vaccines.Staphylococcal load and abscess formation in renal tissue ^(c)Reduction^(e)Number of Antigen ^(a)log₁₀ CFU g⁻¹ ^(b)P-value (log₁₀ CFU g⁻¹)^(d)IgG Titer abscesses ^(b)P-value S. aureus Newman challenge Mock 6.46± 0.25 — — <100 3.7 ± 1.2 — SpA 3.95 ± 0.56 0.0003 2.51 1706 ± 370 2.1 ±1.2 0.3581 SpA-D 4.43 ± 0.41 0.0001 2.03 381 ± 27 1.5 ± 0.8 0.1480SpA-D_(KKAA) 3.39 ± 0.50 <0.000  3.07 5600 ± 801 0.5 ± 0.4 0.0204 S.aureus USA300 (LAC) challenge Mock 7.20 ± 0.24 — — <100 4.0 ± 0.8 — SpA6.81 ± 0.26 0.2819 0.39 476 ± 60 3.3 ± 1.0 0.5969 SpA-D 6.34 ± 0.520.1249 0.86 358 ± 19 2.2 ± 0.6 0.0912 SpA-D_(KKAA) 6.00 ± 0.42 0.01891.20  3710 ± 1147 1.6 ± 0.6 0.0277 SpA_(KKAA) 3.66 ± 0.76 0.0001 3.5410200 ± 2476 1.2 ± 0.5 0.0109 ^(a)Means of staphylococcal loadcalculated as log₁₀ CFU g⁻¹ in homogenized renal tissues 4 daysfollowing infection in cohorts of fifteen to twenty BALB/c mice perimmunization. A representative of three independent and reproducibleanimal experiments is shown. Standard error of the means (±SEM) isindicated. ^(b)Statistical significance was calculated with the unpairedtwo-tailed Students t-test and P-values recorded; P-values < 0.05 weredeemed significant. ^(c)Reduction in bacterial load calculated as log₁₀CFU g⁻¹. ^(d)Means of five randomly chosen serum IgG titers weremeasured prior to staphylococcal infection by ELISA. ^(e)Histopathologyof hematoxylin-eosin stained, thin sectioned kidneys from ten animals;the average number of abscesses per kidney was recorded and averagedagain for the final mean (±SEM).

TABLE 6 Passive immunization of mice with antibodies against protein A.Staphylococcal load and abscess formation in renal tissue ^(d)Reduction^(f)Number of ^(a)Antibody ^(b)log₁₀ CFU g⁻¹ ^(c)P-value (log₁₀ CFU g⁻¹)^(e)IgG Titer abscesses ^(c)P-value Mock 7.10 ± 0.14 — — <100 4.5 ± 0.8— α-SpA-D_(KKAA) 5.53 ± 0.43 0.0016 1.57  466 ± 114 1.9 ± 0.7 0.0235α-SpA_(KKAA) 5.69 ± 0.34 0.0005 1.41 1575 ± 152 1.6 ± 0.5 0.0062^(a)Affinity purified antibodies were injected into the peritonealcavity of BALB/c mice at a concentration of 5 mg · kg⁻¹ twenty-fourhours prior to intravenous challenge with 1 × 10⁷ CFU S. aureus Newman.^(b)Means of staphylococcal load calculated as log₁₀ CFU g⁻¹ inhomogenized renal tissues 4 days following infection in cohorts offifteen BALB/c mice per immunization. A representative of twoindependent and reproducible animal experiments is shown. Standard errorof the means (±SEM) is indicated. ^(c)Statistical significance wascalculated with the unpaired two-tailed Students t-test and P-valuesrecorded; P-values < 0.05 were deemed significant. ^(d)Reduction inbacterial load calculated as log₁₀ CFU g⁻¹. ^(e)Means of five randomlychosen serum IgG titers were measured prior to staphylococcal infectionby ELISA. ^(f)Histopathology of hematoxylin-eosin stained, thinsectioned kidneys from ten animals; the average number of abscesses perkidney was recorded and averaged again for the final mean (±SEM).

Immune response to protein A following staphyloccal infection orSpA_(KKAA) immunization. Following infection with virulent S. aureus,mice do not develop protective immunity against subsequent infectionwith the same strain (Burts et al., 2008)(FIG. 8). The average abundanceof SpA-D_(KKAA) specific IgG in these animals was determined by dot blotas 0.20 μg ml⁻¹ (±0.04) and 0.14 μg ml⁻¹ (±0.01) for strains Newman andUSA300 LAC, respectively (FIG. 4). The minimal concentration of proteinA-specific IgG required for disease protection in SpA_(KKAA) orSpA-D_(KKAA) vaccinated animals (P .0.05 log₁₀ reduction instaphylococcal CFU g⁻¹ renal tissue) was calculated as 4.05 μg ml⁻¹(±0.88). Average serum concentration of SpA-specific IgG in adulthealthy human volunteers (n=16) was 0.21 μg ml⁻¹ (±0.02). Thus, S.aureus infections in mice or humans are not associated with immuneresponses that raise significant levels of neutralizing antibodiesdirected against protein A, which is likely due to the B cellsuperantigen attributes of this molecule. In contrast, the average serumconcentration of IgG specific for diphtheria toxin in human volunteers,0.068 μg ml⁻¹ (±0.20), was within range for protective immunity againstdiphtheria (Behring, 1890; Lagergard et al., 1992).

Clinical S. aureus isolates express protein A, an essential virulencefactor whose B cell surperantigen activity and evasive attributestowards opsono-phagocytic clearance are absolutely required forstaphylococcal abscess formation (Palmqvist et al., 2005; Cheng et al.,2009; Silverman and Goodyear, 2006). Protein A can thus be thought of asa toxin, essential for pathogenesis, whose molecular attributes must beneutralized in order to achieve protective immunity. By generatingnon-toxigenic variants unable to bind Igs via Fcγ or VH₃-Fab domains,the inventors measure here for the first time protein A neutralizingimmune responses as a correlate for protective immunity against S.aureus infection. In contrast to many methicillin-sensitive strains,CA-MRSA isolate USA300 LAC is significantly more virulent (Cheng et al.,2009). For example, immunization of experimental animals with thesurface protein IsdB (Kuklin et al., 2006; Stranger-Jones et al., 2006)raises antibodies that confer protection against S. aureus Newman(Stranger-Jones et al., 2009) but not against USA300 challenge.

Material and Methods

Bacterial strains and growth. Staphylococcus aureus strains Newman andUSA300 were grown in tryptic soy broth (TSB) at 37° C. Escherichia colistrains DH5a and BL21 (DE3) were grown in Luria-Bertani (LB) broth with100 μg ml⁻¹ ampicillin at 37° C.

Rabbit Antibodies. The coding sequence for SpA was PCR-amplified withtwo primers, gctgcacatatggcgcaacacgatgaagctcaac (SEQ ID NO:35) andagtggatccttatgcttgagctttgttagcatctgc (SEQ ID NO:36) using S. aureusNewman template DNA. SpA-D was PCR-amplified with two primers,aacatatgttcaacaaagatcaacaaagc (SEQ ID NO:38) andaaggatccagattcgtttaattttttagc (SEQ ID NO:39). The sequence forSpA-D_(KKAA) was mutagenized with two sets of primerscatatgttcaacaaagataaaaaaagcgccttctatgaaatc (SEQ ID NO:42) andgatttcatagaaggcgctttttttatctttgttgaacatatg (SEQ ID NO:43) for Q9K, Q10Kas well as cttcattcaaagtcttaaagccgccccaagccaaagcactaac (SEQ ID NO:40)and gttagtgctttggcttggggcggctttaagactttgaatgaag (SEQ ID NO:41) forD36A,D37A. The sequence of SpA_(KKAA) was synthesized by Integrated DNATechnologies, Inc. PCR products were cloned into pET-15b generatingN-terminal His₆ tagged recombinant protein. Plasmids were transformedinto BL21(DE3). Overnight cultures of transformants were diluted 1:100into fresh media and grown at 37° C. to an OD₆₀₀ 0.5, at which pointcultures were induced with 1 mM isopropyl β-D-1-thiogalatopyranoside(IPTG) and grown for an additional three hours. Bacterial cells weresedimented by centrifugation, suspended in column buffer (50 mMTris-HCl, pH 7.5, 150 mM NaCl) and disrupted with a French pressure cellat 14,000 psi. Lysates were cleared of membrane and insoluble componentsby ultracentrifugation at 40,000×g. Proteins in the soluble lysate weresubjected to nickel-nitrilotriacetic acid (Ni-NTA, Qiagen) affinitychromatography. Proteins were eluted in column buffer containingsuccessively higher concentrations of imidazole (100-500 mM). Proteinconcentrations were determined by bicinchonic acid (BCA) assay (ThermoScientific). For antibody generation, rabbits (6 month old New-Zealandwhite, female, Charles River Laboratories) were immunized with 500 μgprotein emulsified in Complete Freund's Adjuvant (Difco) by subscapularinjection. For booster immunizations, proteins emulsified in IncompleteFreund's Adjuvant and injected 24 or 48 days following the initialimmunization. On day 60, rabbits were bled and serum recovered.

Antibody Isolation. Purified antigen (5 mg protein) was covalentlylinked to HiTrap NHS-activated HP columns (GE Healthcare).Antigen-matrix was used for affinity chromatography of 10-20 ml ofrabbit serum at 4° C. Charged matrix was washed with 50 column volumesof PBS, antibodies eluted with elution buffer (1 M glycine, pH 2.5, 0.5M NaCl) and immediately neutralized with 1 M Tris-HCl, pH 8.5. Purifiedantibodies were dialyzed overnight against PBS at 4° C.

F(ab)₂ fragments. Affinity purified antibodies were mixed with 3 mg ofpepsin at 37° C. for 30 minutes. The reaction was quenched with 1 MTris-HCl, pH 8.5 and F(ab)₂ fragments were affinity purified withspecific antigen-conjugated HiTrap NHS-activated HP columns. Purifiedantibodies were dialyzed overnight against PBS at 4° C., loaded ontoSDS-PAGE gel and visualized with Coomassie Blue staining

Active and passive immunization. BALB/c mice (3 week old, female,Charles River Laboratories) were immunized with 50 μg protein emulsifiedin Complete Freund's Adjuvant (Difco) by intramuscular injection. Forbooster immunizations, proteins were emulsified in Incomplete Freund'sAdjuvant and injected 11 days following the initial immunization. On day20 following immunization, 5 mice were bled to obtain sera for specificantibody titers by enzyme-linked immunosorbent assay (ELISA).

BALB/c mice were immunized by intramuscular injection and boosted withthe same antigen in Alum after 11 and 25 days. On day 34, mice were bledto obtain serum for specific antibody titers. Affinity purifiedantibodies were injected into the peritoneal cavity of BALB/c miceeither 24 hours or 4 hours prior to sub-lethal or lethal challenge,respectively. Animal blood was collected via periorbital vein punctureand antigen specific serum antibody titers measured by ELISA.

Mouse renal abscess. Overnight cultures of S. aureus Newman or USA300(LAC) were diluted 1:100 into fresh TSB and grown for 2 hours at 37° C.Staphylococci were sedimented, washed and suspended PBS at OD₆₀₀ of 0.4(˜1×10⁸ CFU ml⁻¹). Inocula were quantified by spreading sample aliquotson TSA and enumerating colonies formed. BALB/c mice (6 week old, female,Charles River Laboratories) were anesthetized via intraperitonealinjection with 100 mg ml⁻ketamine and 20 mg ml⁻¹ xylazine per kilogramof body weight. Mice were infected by retro-obital injection with 1×10⁷CFU of S. aureus Newman or 5×10⁶ CFU of S. aureus USA300. On day 4following challenge, mice were killed by CO₂ inhalation. Both kidneyswere removed, and the staphylococcal load in one organ was analyzed byhomogenizing renal tissue with PBS, 1% Triton X-100. Serial dilutions ofhomogenate were spread on TSA and incubated for colony formation. Theremaining organ was examined by histopathology. Briefly, kidneys werefixed in 10% formalin for 24 hours at room temperature. Tissues wereembedded in paraffin, thin-sectioned, stained with hematoxylin-eosin,and inspected by light microscopy to enumerate abscess lesions. Allmouse experiments were performed in accordance with the institutionalguidelines following experimental protocol review and approval by theInstitutional Biosafety Committee (IBC) and the Institutional AnimalCare and Use Committee (IACUC) at the University of Chicago.

Mouse infection. Staphylococci were used to infect anesthetized mice byretro-orbital injection (1×10⁷ CFU of S. aureus Newman, 5×10⁶ CFU of S.aureus USA300 or 3×10⁷ CFU of S. aureus Mu50). On day 4, 15 or 30, micewere killed, kidneys removed, and homogenized tissue spread on agar forcolony formation. Organ tissue was also thin-sectioned, stained withhematoxylin-eosin, and viewed by microscopy. Animal experiments wereperformed in accordance with the institutional guidelines followingexperimental protocol review and approval by the Institutional BiosafetyCommittee (IBC) and the Institutional Animal Care and Use Committee(IACUC) at the University of Chicago.

For lethal challenge experiments, BALB/c mice (cohorts of 8-10 animalsper experiment) were injected with a suspension of 2−6×10⁸ CFU of S.aureus Newman or its isogenic Δspa variant into the peritoneal cavity.Animal survival was monitored over a period of 15 days and statisticalsignificance of survival data analyzed with the log-rank test.

Protein A binding. For human IgG binding, Ni-NTA affinity columns werepre-charged with 200 μg of purified proteins (SpA, SpA-D, SpA-D_(KKAA),and SrtA) in column buffer. After washing, 200 μg of human IgG (Sigma)was loaded onto the column. Protein samples were collected from washesand elutions and subjected to SDS-PAGE gel electrophoresis, followed byCoomassie Blue staining Purified proteins (SpA, SpA_(KKAA), SpA-D andSpA-D_(KKAA)) were coated onto MaxiSorp ELISA plates (NUNC) in 0.1Mcarbonate buffer (pH 9.5) at 1 μg ml⁻¹ concentration overnight at 4° C.Plates were next blocked with 5% whole milk followed by incubation withserial dilutions of peroxidase-conjugated human IgG, Fc or F(ab)₂fragments for one hour. Plates were washed and developed using OptEIAELISA reagents (BD). Reactions were quenched with 1 M phosphoric acidand A₄₅₀ readings were used to calculate half maximal titer and percentbinding.

von Willebrand Factor (vWF) binding assays. Purified proteins (SpA,SpA_(KKAA), SpA D and SpA-D_(KKAA)) were coated and blocked as describedabove. Plates were incubated with human vWF at 1 μg ml⁻¹ concentrationfor two hours, then washed and blocked with human IgG for another hour.After washing, plates were incubated with serial dilution ofperoxidase-conjugated antibody directed against human vWF for one hour.Plates were washed and developed using OptEIA ELISA reagents (BD).Reactions were quenched with 1 M phosphoric acid and A₄₅₀ readings wereused to calculate half maximal titer and percent binding. For inhibitionassays, plates were incubated with affinity purified F(ab)₂ fragmentsspecific for SpA-D_(KKAA) at 10 μg ml⁻¹ concentration for one hour priorto ligand binding assays.

Splenocyte apoptosis. Affinity purified proteins (150 μg of SpA, SpA-D,SpA_(KKAA), and SpA-D_(KKAA)) were injected into the peritoneal cavityof BALB/c mice (6 week old, female, Charles River Laboratories). Fourhours following injection, animals were killed by CO₂ inhalation. Theirspleens were removed and homogenized. Cell debris were removed usingcell strainer and suspended cells were transferred to ACK lysis buffer(0.15 M NH₄Cl, 10 mM KHCO₃, 0.1 mM EDTA) to lyse red blood cells. Whiteblood cells were sedimented by centrifugation, suspended in PBS andstained with 1:250 diluted R-PE conjugated anti-CD19 monoclonal antibody(Invitrogen) on ice and in the dark for one hour. Cells were washed with1% FBS and fixed with 4% formalin overnight at 4° C. The following day,cells were diluted in PBS and analyzed by flow cytometry. The remainingorgan was examined for histopathology. Briefly, spleens were fixed in10% formalin for 24 hours at room temperature. Tissues were embedded inparaffin, thin-sectioned, stained with the Apoptosis detection kit(Millipore), and inspected by light microscopy.

Antibody quantification. Sera were collected from healthy humanvolunteers or BALB/c mice that had been either infected with S. aureusNewman or USA300 for 30 days or that had been immunized withSpA-D_(KKAA)/SpA_(KKAA) as described above. Human/mouse IgG (JacksonImmunology Laboratory), SpA_(KKAA), and CRM₁₉₇ were blotted ontonitrocellulose membrane. Membranes were blocked with 5% whole milk,followed by incubation with either human or mouse sera. IRDye 700DXconjugated affinity purified anti-human/mouse IgG (Rockland) was used toquantify signal intensities using the Odyssey™ infrared imaging system(Li-cor). Experiments with blood from human volunteers involvedprotocols that were reviewed, approved and performed under regulatorysupervision of The University of Chicago's Institutional Review Board(IRB).

Statistical Analysis. Two tailed Student's t tests were performed toanalyze the statistical significance of renal abscess, ELISA, and B cellsuperantigen data. Animal survival data were analyzed with the log-ranktest (Prism).

Example 3 Coagulases of Staphylococcus Aureus Contribute to AbscessesFormation and Function as Protective Antigens

All clinical S. aureus isolates display coagulase activity—the clottingof blood or plasma through non-proteolytic activation of prothrombin tocleave fibrinogen. The inventors identified prothrombin, fibrinogen,coagulase (Coa) and von Willebrand-factor binding protein (vWbp) instaphylococcal abscess lesions of infected mice. Secreted Coa and vWbpboth contributed to S. aureus Newman coagulase activity, therebyenabling abscess formation as well as lethal disease in mice. Antibodiesraised against purified Coa or vWbp specifically block association ofthe corresponding polypeptide with prothrombin and fibrinogen. Coa- andvWbp-specific antibodies, whether raised by active or passiveimmunization, prevented abscess formation and mortality of mice infectedwith staphylococci.

Results

Localization of coagulase and coagulation factors in staphylococcalabscesses. Previous work established the mouse renal abscess model,whereby 1×10⁷ CFU of the human clinical isolate S. aureus Newman (Babaet al., 2007) are injected into the blood stream of BALB/c mice (Albuset al., 1991). Forty-eight hours following infection, mice developdisseminated abscesses in multiple organs, detectable by lightmicroscopy of hematoxylin-eosin stained, thin-sectioned kidney tissueinitially as an accumulation of polymorphonuclear leukocytes (PMNs) withfew bacteria (Cheng et al., 2009). By day 5 of infection, abscessesincrease in size and enclose a central population of staphylococci(staphylococcal abscess community—SAC), surrounded by a layer ofeosinophilic, amorphous material (the pseudocapsule) and a large cuff ofPMNs (Cheng et al., 2009). Histopathology reveals massive necrosis ofPMNs in proximity to the staphylococcal nidus at the center of abscesslesions as well as a mantle of healthy phagocytes. At later timeintervals, SACs increase and abscesses rupture, releasing necroticmaterial and staphylococci into the bloodstream. A new round of abscessformation is initiated, eventually precipitating a lethal outcome ofinfections (Cheng et al., 2009).

To localize coagulases in abscess lesions, kidneys of mice that had beeninfected for 5 days with S. aureus Newman were thin-sectioned andstained by immuno-histochemistry with affinity purified Coa- orvWbp-specific rabbit antibodies (FIG. 10). The inventors observedintense Coa staining in the pseudocapsule surrounding SACs and in theperiphery of abscess lesions, i.e., the fibrin capsule borderinguninfected tissue. vWbp staining occurred throughout abscess lesions,but also with accumulation at the periphery. Prothrombin specificantibodies revealed staining of the zymogen in the pseudocapsule and inthe periphery, whereas fibrinogen/fibrin specific staining occurredthroughout abscess lesions. Together these data indicate that theeosinophilic pseudocapsule of staphylococcal abscesses harborsprothrombin and fibrinogen, which co-localize with Coa. At the peripheryof abscess lesions, Coa, vWbp, prothrombin and fibrinogen/fibrin areco-localized. These observations prompted further investigation in towhether Coa and vWbp are crucial contributors to the establishment ofabscesses by triggering prothombin-mediated conversion of fibrinogen tofibrin.

Staphylococcus aureus coa and vWbp contribute to the clotting of mouseblood. The coa and/or vWbp genes on the chromosome of S. aureus Newmanwere deleted by allelic replacement using pKOR1 technology (Bae andSchneewind, 2005). Two complementing plasmids, pcoa and pvWbp, weregenerated by cloning coa or vWbp structural genes as well as theirupstream promoter sequences into pOS1 (Schneewind et al., 1993).Plasmids were electroporated into staphylococci and their continuedreplication selected on media supplemented with chloramphenicol(Schneewind et al., 1992). When probed for coagulases with specificantibodies, the inventors observed Coa secretion by the wild-type aswell as the ΔvWbp strain, but not by Δcoa or Δcoa/ΔvWbp variants (FIG.11). The phenotypic defect of Δcoa and Δcoa/ΔvWbp mutants was restoredby electroporation with pcoa but not by pvWbp (FIG. 11). Similarly,secretion of vWbp was observed in S. aureus Newman (wild-type) as wellas Δcoa mutant cultures, but not in ΔvWbp or Δcoa/ΔvWbp variants (FIG.11). This defect was restored by electroporation with pvWbp, but not bypcoa.

Clotting of blood is effectively inhibited by hirudin (lepirudin)(Harvey et al., 1986), a 65 residue peptide from leech that forms a 1:1complex with thrombin, thereby blocking proteolytic conversion offibrinogen to fibrin (Markwardt, 1955). Inoculation of freshlepirudin-treated mouse blood with S. aureus Newman triggered clottingin less than 12 hours, whereas mock infected blood remained withoutclots for more than 48 hours (FIG. 11C). Using this assay, it wasobserved that staphylococcal variants lacking coagulase activitydisplayed delays in clotting time, Δcoa 36 hours and ΔvWbp 24 hours(FIG. 11C). The double mutant, Δcoa/ΔvWbp, was unable to clot mouseblood. These defects were complemented by electroporation with plasmidspvWbp as well as pcoa. Taken together, these data indicate that the twocoagulases, Coa and vWbp, contribute to the ability of S. aureus Newmanto clot mouse blood (FIG. 11C).

Coa and vWbp are required for staphylococcal survival in blood, abscessformation and lethal bacteremia in mice. To analyze the virulencecontributions of coagulases, the inventors first examined staphylococcalsurvival in lepirudin-treated blood. Wild-type strain S. aureus Newmanwas not killed in mouse blood, however isogenic variants lacking Coa,i.e. Δcoa and Δcoa/ΔvWbp, each displayed a significant reduction in CFUafter 30 min incubation. This defect in survival was restored by pcoa,but not by pvWbp, suggesting that only Coa is required forstaphylococcal survival in mouse blood.

Staphylococcal bacteremia is a frequent cause of human mortality inhospital settings (Klevens et al., 2007). The inventors sought toascertain whether coagulases are required for lethal challenge of BALB/cmice, following intravenous injection of 1×10⁸ CFU S. aureus Newman. Allanimals infected with the wild-type parent strain Newman succumbed toinfection within 24 hours (FIG. 12B). Animals infected with singlemutants, Δcoa or ΔvWbp, each displayed a short but statisticallysignificant delay in time-to-death (FIG. 12B). The double mutant strainwas significantly more impaired than mutants with single deletions andanimals infected with the Δcoa/ΔvWbp strain displayed the largestreduction in virulence as compared to the wild-type (FIG. 12B).

The inventors next analyzed abscess formation in renal tissues ofinfected mice and observed that Δcoa variants were impaired in theirability to form abscesses by day 5 and 15 of infection (Table 7, FIGS.12G, 12I)). The ΔvWbp mutant continued to form abscesses, although thebacterial load, the overall size of staphylococcal abscess communitiesand the amount of immune cell infiltrates were reduced in these variants(Table 7, FIGS. 12D, 12F)). Mutants in coagulase are slightly moreattenuated in virulence than those in vWbp, as Δcoa has lower abscessformation and bacterial load by day 15. However, the Δcoa/ΔvWbp doublemutants markedly incapacitated in their ability to form abscesses andpersist in infected tissues (Table 7, FIGS. 12H, 12K)). Thus, bothcoagulase and von Willebrand factor binding protein are important forstaphylococcal survival in the host, whether in the bloodstream or endorgan tissues.

TABLE 7 Virulence of S. aureus Newman coa, vWbp, and coa/vWbp mutantsAbscess formation in kidney tissue* Staphylococcal load in kidneytissue* ^(f)Number of ^(a)log₁₀ CFU g⁻¹ of ^(b)Significance^(c)Reduction in ^(e)Surface abscesses per ^(g)Significance Strainkidney tissue (P-value) ^(a)log₁₀ CFU g⁻¹ abscesses (%) kidney (P-value)Day 5 analysis of staphylococcal load and abscess formation PBS 6.034 ±0.899 — — 75 2.333 ± 0.623 — Coa 5.538 ± 0.560 0.3750 0.492 38 1.111 ±0.389 0.1635 vWbp 5.247 ± 0.311 0.0859 0.783 56 1.750 ± 0.650 0.6085coa/vWbp 4.908 ± 0.251 0.0044 1.395 25 0.750 ± 0.342 0.0786 Day 15analysis of staphylococcal load and abscess formation PBS 5.380 ± 0.294— — 81 3.000 ± 1.234 — Coa 4.023 ± 0.324 0.0077 1.357 44 1.400 ± 0.4520.1862 vWbp 5.140 ± 0.689 0.0688 0.240 50 1.625 ± 0.298 0.2974 coa/vWbp3.300 ± 0.552 0.0056 2.080 20 0.556 ± 0.154 0.0341 *BALB/c mice (n =18-20) were injected into the peritoneum with 100 μl each of affinitypurified rabbit antibodies against vWbp (α-vWbp), Coa (α-Coa) or vWbpand Coa (α-vWbp/Coa) on day 0. Twenty four hours later, animals wereexamined for IgG antibody titers in serum and were challenged byintravenous inoculation with 1 × 10⁷ colony forming units (CFU) S.aureus Newman or mutants thereof. Five or fifteen days later, animalswere killed and both kidneys removed. One kidney was fixed informaldehyde, embedded in paraffin, thin sectioned, hemaotoxylin-eosinstained and four saggital sections per kidney were analyzed for abscessformation. The other kidney was homogenized in PBS buffer, homogenatespread on agar medium for colony formation, and staphylococcal loadenumerated as CFU. ^(a)Means of staphylococcal load calculated as log₁₀CFU g⁻¹ in homogenized renal tissues 4 days following infection incohorts of eighteen to twenty BALB/c mice per immunization. Standarderror of the means (±SEM) is indicated. ^(b)Statistical significance wascalculated with the unpaired two-tailed Students t-test and P-valuesrecorded; P-values < 0.05 were deemed significant. ^(c)Reduction inbacterial load calculated as log₁₀ CFU g⁻¹. ^(e)Abscess formation inkidney tissues four days following infection was measured by macroscopicinspection (% positive) ^(f)Histopathology of hematoxylene-eosinstained, thin sectioned kidneys from ten animals; the average number ofabscesses per kidney was recorded and averaged again for the final mean(±SEM). ^(g)Statistical significance was calculated with the unpairedtwo-tailed Students t-test and P-values recorded; P-values < 0.05 weredeemed significant.

Antibodies against coagulases and their effect on blood clotting inducedduring staphylococcal infection. Recombinant His₆-Coa and His₆-vWbp werepurified by affinity chromatography on Ni-NTA (FIG. 13A), emulsified inadjuvant and injected into rabbits to raise specific antibodies thatwere purified on affinity matrices harboring recombinant protein.Antibodies directed against Coa preferentially bound to Coa, not to vWbp(FIG. 13B). The reciprocal was true for antibodies directed against vWbp(FIG. 13B). When added to lepirudin-treated mouse blood infected with S.aureus Newman, the inventors observed that antibodies directed againstCoa, vWbp or Coa and vWbp each blocked the coagulation of blood (FIG.13C). As controls, mock treated samples or the irrelevant V10 antibody(which provides protection against Yersinia pestis type III injection(DeBord et al., 2006)) had no effect (FIG. 13C).

To examine the role of antibodies on isolated Coa or vWbp, the inventorspurified recombinant, functionally active proteins (Friedrich et al.,2003) that were then added to lepirudin treated mouse blood. Coa or vWbptreated mouse blood coagulated in less than 30 minutes (FIG. 13D). As acontrol, mock (PBS) or treatment with irrelevant V10 antibody did notaffect clotting. Antibodies directed against Coa or vWbp delayedclotting of mouse blood treated with recombinant proteins and thisoccurred even for the cross-reacting homologous factor (FIG. 13D).Minimal cross reactivity of the antibodies was observed by ELISA andwestern blot, yet there is cross inhibition of function.

Antibodies that block association between coagulases and prothrombin orfibrinogen. Surface plasmon resonance (SPR) was used to investigate howaCoa and avWbp antibodies interfere with the physiological functions ofcoagulases. Prothrombin was immobilized on a CM5 chip. Flowing purifiedCoa over the sample, a dissociation constant K_(D) 28 nM was caluclated,a measurement that is commensurate with other reports in the literature(Friedrich et al., 2003). The addition of aCoa led to aconcentration-dependent decrease in response signal for the formation ofprothrombin•Coa, indicating that these antibodies block association ofCoa with prothrombin (FIG. 14A). SPR further confirmed associationbetween coagulase and fibrinogen (K_(D) 93.1 nM, FIG. 14B). Uponpre-incubation with aCoa, the inventors observed a dramatic decrease inthe binding of Coa to fibrinogen (FIG. 14B). Taken together, theseresults indicate that antibodies directed against Coa block theassociation of this molecule with blood coagulation factors.

Purified vWbp displayed strong affinity for prothrombin (K_(D) 38.4 nM,FIG. 13C) and fibrinogen (484 nM, FIG. 13D), the latter of which hadhitherto not been appreciated (Kroh et al., 2009). Further,pre-incubation with antibodies raised against vWbp blocked theassociation between vWbp and prothrombin or fibrinogen in adose-dependent manner (FIGS. 13C, 13D). These findings support resultsfrom the blood coagulation assays, demonstrating that specificpolyclonal antibodies can block the interaction between Coa or vWbp andspecific components of the coagulation cascade (FIG. 12).

To test whether antibodies specific for coagulases block the conversionof fibrinogen to fibrin, the ability of prothrombin•coagulase complexesto cleave S-2238 was measured, a surrogate for the cleavage offibrinogen to fibrin (FIGS. 14E, 14F). Addition of specific antibodiesto prothrombin•Coa or prothrombin•vWbp reduced the ability of thesecomplexes to convert substrate to product. Further, cross-inhibition ofcoagulase-specific antibodies was observed, where the addition ofcross-reacting antibodies caused a reduction in activity of theprothrombin•vWbp complex. These data suggest that specific antibodiesdirected against Coa or vWbp neutralize the pathophysiological effect ofthe secreted product to which they bind.

Antibodies against coagulases provide protection against staphylococcaldisease. IgG type antibodies specific for Coa or vWbp were isolated fromrabbit serum by chromatography over an affinity column, generated bycovalent crosslinking of the antigen to CNBr sepharose. The inventorsattempted to perturb staphylococcal pathogenesis by administration ofneutralizing antibodies, directed against Coa and/or vWbp. Mice wereadministered rabbit antibodies and challenged with a lethal dose of S.aureus strain Newman. Injection of Coa or vWbp specific antibodiessignificantly prolonged murine survival (FIG. 15).

To test antibody reagents for possible vaccine protection against lethalbacteremia, affinity purified IgG (5 mg kg⁻¹ body weight) were injectedinto the peritoneal cavity of mice. Twenty-four hours later, animalswere injected with a suspension of 1×10⁸ CFU S. aureus Newman in PBSinto the retro-orbital plexus. Monitoring animals over time, theinventors observed that antibodies directed against vWbp (avWbp) led toincreased time-to-death and to 10% survival, as compared to animals thathad received irrelevant αV10 antibodies and died within 12-48 hours(FIG. 15). Antibodies against Coa (αCoa) further increased thetime-to-death of passively immunized mice (FIG. 15). A mixture of bothantibodies (αCoa/αvWbp) did not generate a statistically significantimprovement in survival or time-to-death over αCoa antibodies.

To examine the passive immunization for protection againststaphylococcal abscess formation, purified antibodies (5 mg kg-1 bodyweight) were injected into the peritoneal cavity of mice and abscessformation was monitored for five days after intravenous challenge with1×10⁷ CFU S. aureus Newman. Antibodies against vWbp did not lead to asignificant reduction in staphylococcal load or in the number ofinflammatory lesions (Table 8), although the observed lesions harboredsmaller abscess communities and reduced PMN infiltrates as compared tomock immunized mice (FIG. 16). Antibodies against coagulase reduced thestaphylococcal load (P=0.042) as well as the number of inflammatorylesions (P=0.039); abscess lesions with staphylococcal communities atthe nidus of large PMN infiltrates were not detected (FIG. 16 and Table8). Animals that received both antibodies, αWbp and αCoa, displayed aneven greater reduction in staphylococcal load (P=0.013) and a reductionin the abundance of inflammatory lesions (P=0.0078) (Table 8). Together,these data indicate that antibodies against coagulases, administered bypassive immunization, protect mice against abscess formation and enableclearance of the invading pathogen from host tissues. Antibodies againstvWbp contribute relatively little to vaccine protection, in agreementwith the finding that vWbp does not play the same critical role as Coaduring the pathogenesis of S. aureus infections in mice (Table 8).

TABLE 8 Passive immunization of mice with rabbit antibodies against Coaand/or vWbp Abscess formation in kidney tissue* Purified Staphylococcalload in kidney tissue* ^(f)Number of Rabbit ^(a)log₁₀ CFU g⁻¹ of^(b)Significance ^(c)Reduction in ^(e)Surface abscesses per^(g)Significance Antibody kidney tissue (P-value) ^(a)log₁₀ CFU g⁻¹^(d)IgG Titer abscesses (%) kidney (P-value) Mock 5.86 ± 0.29 — — <10075 4.6 ± 1.4 — α-vWbp 5.25 ± 0.36 0.3554 0.60 1,100 ± 200 39 1.4 ± 0.50.0592 α-Coa 4.68 ± 0.47 0.0420 1.18 1,300 ± 250 20 1.2 ± 0.7 0.0396α-vWbp/Coa 4.29 ± 0.52 0.0130 1.53 1,000 ± 300 25 0.3 ± 0.2 0.0078*BALB/c mice (n = 18-20) were injected into the peritoneum with 100 μleach of affinity purified rabbit antibodies against vWbp (α-vWbp), Coa(α-Coa) or vWbp and Coa (α-vWbp/Coa) on day 0. Twenty four hours later,animals were examined for IgG antibody titers in serum and werechallenged by intravenous inoculation with 1 × 10⁷ colony forming units(CFU) S. aureus Newman. Five days later, animals were killed and bothkidneys removed. One kidney was fixed in formaldehyde, embedded inparaffin, thin sectioned, hemaotoxylin-eosin stained and four saggitalsections per kidney were analyzed for abscess formation. The otherkidney was homogenized in PBS buffer, homogenate spread on agar mediumfor colony formation, and staphylococcal load enumerated as CFU.^(a)Means of staphylococcal load calculated as log₁₀ CFU g⁻¹ inhomogenized renal tissues 4 days following infection in cohorts ofeighteen to twenty BALB/c mice per immunization. Standard error of themeans (±SEM) is indicated. ^(b)Statistical significance was calculatedwith the unpaired two-tailed Students t-test and P-values recorded;P-values < 0.05 were deemed significant. ^(c)Reduction in bacterial loadcalculated as log₁₀ CFU g⁻¹. ^(e)Abscess formation in kidney tissuesfour days following infection was measured by macroscopic inspection (%positive) ^(f)Histopathology of hematoxylene-eosin stained, thinsectioned kidneys from ten animals; the average number of abscesses perkidney was recorded and averaged again for the final mean (±SEM).^(g)Statistical significance was calculated with the unpaired two-tailedStudents t-test and P-values recorded; P-values < 0.05 were deemedsignificant.

Coagulases function as protective antigens for staphylococcalinfections. Poly-histidine tagged CoA and vWbp were purified from E.coli and used as subunit vaccine antigens. Proteins (100 μg emulisifiedin CFA or IFA) were injected into naïve BALB/c mice on day 0 (CFA) or 11(IFA). Animals were challenged on day 21 by intravenous inoculation ofS. aureus Newman. Five control animals were bled at the time ofchallenge and serum antibody titers against vaccine antigens weredetermined by ELISA (Table 9). Animals were killed five or fifteen daysfollowing challenge staphylococcal load and histopathology of abscesslesions were analyzed. Immunization with Coa reduced the bacterial loadby day 5 (P=0.03, PBS mock vs. Coa) and day 15 (P=4.286×10⁻⁵, PBS mockvs. Coa, see Table 9). Coa vaccination also diminished the number ofinfectious lesions that formed in kidney tissues, mock vs. Coa, P=0.03(day 5) and P=0.0522 (day 15) (Table 9). Of note, none of theCoa-immunized mice developed typical abscess lesions (FIG. 17). Onoccasion small accumulations of PMNs that were not associated withstaphylococcal abscess communities were observed (FIG. 17). Immunizationwith vWbp did not significantly reduce staphylococcal load on day 5(P=0.39, PBS mock vs. vWbp) or on day 15 (P=0.09, PBS mock vs. vWbp).The total number of inflammatory lesions was not reduced. Nevertheless,the architecture of abscesses had changed following immunization withvWbp. Staphylococcal communities were not detected at the center ofabscesses and instead PMN infiltrations were observed (FIG. 17). Thecombination vaccine, vWbp-Coa, further reduced the number ofinflammatory cells in kidney tissues and infected animals did notdisplay abscess lesions on day 5 or 15 (Table 9).

TABLE 9 Active immunization of mice with Coa and/or vWbp Staphylococcalload in kidney tissue* Abscess formation in kidney tissue* Purified^(c)Reduction ^(e)Surface ^(f)Number of Vaccine ^(a)log₁₀ CFU g⁻¹ of^(b)Significance in ^(a)log₁₀ abscesses abscesses ^(g)SignificanceAntigen kidney tissue (P-value) CFU g⁻¹ ^(d)IgG Titer (%) per kidney(P-value) Day 5^(h) Mock 5.75 ± 0.42 — — <100 56 1.3 ± 0.3 — vWbp 4.94 ±0.46 0.1413 0.81 14,000 ± 5,000 45 1.8 ± 0.5 0.39 Coa 4.86 ± 0.50 0.14170.88 19,000 ± 4,000 25 0.3 ± 0.3 0.03 vWbp/Coa 4.84 ± 0.38 0.1195 0.90 7,000 ± 1,500 25 0.3 ± 0.3 0.03 Day 15^(i) Mock 6.68 ± 0.22 — — <100 756.0 ± 1.9 — vWbp 3.41 ± 0.47 0.4503 3.27 14,000 ± 5,000 20 1.8 ± 1.10.09 Coa 3.43 ± 0.54 0.1681 3.25 19,000 ± 4,000 20 1.2 ± 0.8 0.05vWbp/Coa 3.79 ± 0.37 0.0263 2.89  7,000 ± 1,500 30 0.7 ± 0.5 0.01*BALB/c mice (n = 18-20) were injected with 100 μg each of purifiedvWbp, Coa or vWbp and Coa emulsified in CFA on day 0 and boosted withthe same antigen emulsified in IFA on day 11. On day 20, animals wereexamined for IgG antibody titers and on day 21 animals were challengedby intravenous inoculation with either 1 × 10⁷ colony forming units(CFU) S. aureus Newman. On day 25, animals were killed and both kidneysremoved. One kidney was fixed in formaldehyde, embedded in paraffin,thin sectioned, hemaotoxylin-eosin stained and four saggital sectionsper kidney were analyzed for abscess formation. The other kidney washomogenized in PBS buffer, homogenate spread on agar medium for colonyformation, and staphylococcal load enumerated as CFU. ^(a)Means ofstaphylococcal load calculated as log₁₀ CFU g⁻¹ in homogenized renaltissues 4 days following infection in cohorts of eighteen to twentyBALB/c mice per immunization. Standard error of the means (±SEM) isindicated. ^(b)Statistical significance was calculated with the unpairedtwo-tailed Students t-test and P-values recorded; P-values < 0.05 weredeemed significant. ^(c)Reduction in bacterial load calculated as log₁₀CFU g⁻¹. ^(d)Means of five randomly chosen serum IgG titers weremeasured prior to staphylococcal infection by ELISA with SpA-D_(KKAA)antigen ^(e)Abscess formation in kidney tissues four days followinginfection was measured by macroscopic inspection (% positive)^(f)Histopathology of hematoxylene-eosin stained, thin sectioned kidneysfrom ten animals; the average number of abscesses per kidney wasrecorded and averaged again for the final mean (±SEM). ^(g)Statisticalsignificance was calculated with the unpaired two-tailed Students t-testand P-values recorded; P-values < 0.05 were deemed significant.^(h)Analysis of mice 5 days following infection with S. aureus Newman.^(i)Analysis of mice 15 days following infection with S. aureus Newman.

Materials and Methods

Bacterial strains and growth of cultures. Staphylococci were cultured ontryptic soy agar or broth at 37° C. E. coli strains DH5a and BL21(DE3)(Studier et al., 1990) were cultured on Luria agar or broth at 37° C.Ampicillin (100 μg/ml) and chloramphenicol (10 μg/ml) were used forpET15b (Studier et al., 1990) and pOS1 (Schneewind et al., 1993) plasmidselection, respectively.

Generation of mutants. DNA sequences 1 kb upstream and downstream of coaand vWbp were PCR amplified using the primers attB1_Coa, Coa1_BamHI,Coa2_BamHI, attbB2_Coa and attB1_vWF, vWF1_BamHI, vWF2_BamHI, attbB2 vWF(Table 10). The fragments were exchanged onto pKOR1 using the BP clonaseII kit (Invitrogen) (Bae and Schneewind, 2005). These vectors wereelectroporated into S. aureus Newman and subjected to temperature shiftinduced allelic exchange to generate the corresponding deletion (Bae andSchneewind, 2005). Mutants were verified by PCR amplification of thegene locus, DNA sequencing, and immunoblot analysis.

To generate complementing plasmids, the primers Coa_promoter_BamHI_F,Coa_out_PstI_R, vWbp_promoter_BamHI_F, vWbp_out_PstI_R (Table 10) weredesigned to include the upstream promoter region of vWbp or coa and theamplified regions were cloned into pOS1. These plasmids were verified bysequencing and then electroporated into staphylococcal strains. Forimmunoblot analysis, overnight cultures of staphylococci grown intryptic soy broth (Difco) were refreshed 1:100 and grown with shaking at37° C. until they reached OD₆₀₀ of 0.4. One ml samples of each culturecentrifuged at 13,000×g for 10 min in a table top centrifuge and thesupernatant was recovered. Trichloroacetic acid, 75 μl of 100% w/vsolution, was added and samples were incubated on ice for 10 min,followed by centrifugation and wash with 1 ml ice-cold 100% acetone.Samples were air dried overnight and solubilized in 50 μl sample buffer(4% SDS, 50 mM Tris-HCl, pH8, 10% glycerol, and bromophenol blue).

Blood survival assay and blood coagulation. Overnight cultures ofstaphylococcal strains were diluted 1:100 into fresh TSB and grown at37° C. until they reached an OD₆₀₀ 0.4. One ml of culture wascentrifuged, and staphylococci washed and suspended in 10 ml of sterilePBS to generate a suspension of 1×10⁷ CFU/ml. Whole blood from naïve 6week old Balb/c mice was collected and REFLUDAN™ (lepirudin, Bayer) wasadded to a final concentration 50 μg/ml. 450 μL blood was aliquoted intoa 1 ml eppendorf tube and mixed with 50 μl bacterial sample (1×10⁵CFU/ml). Samples were incubated at 37° C. with slow rotation. 100 μlaliquots were removed at times 0 min and 30 min, mixed 1:1 with 2%saponin/PBS and incubated on ice for 30 minutes. Five 1:10 serialdilutions were prepared and 10 μl aliquots spread on TSA agar for colonyformation and enumeration.

To assess bacterial blood coagulating activity, 10 μl of the above stockbacterial culture was added to 100 μl of anti-coagulated mouse blood ina sterile plastic test tube (BD falcon) to achieve an end concentrationof 1×10⁵ CFU/ml. For antibody perturbation, an additional 10 ul of PBScontaining 3×10⁻⁵ Mol of antibody was added to the mixture. To assessrecombinant proteins, 10 μl of protein in PBS buffer added to an endconcentration of 50 μM. Test tubes were incubated at room temperatureand blood liquidity or coagulation was verified by tipping the tubes to45° angles in timed intervals.

Protein purification. For vaccination studies, full-length codingsequence of mature Coa or vWbp was cloned into pET15b vector using theprimers Coa_foward_XhoI, Coa_reverse_BamHI, vWbp_forward_XhoI,vWbp_reverse_BamHI (Table 10) to obtain His₆-Coa and His₆-vWbp. E. coliBL21(DE3) harboring expression vectors were grown at 37° C. and inducedwith 1 mM IPTG after two hours. Four hours after induction, cells werecentrifuged at 6,000×g, supended in 1×column buffer (0.1 M Tris-HCl pH7.5, 0.5 M NaCl) and lysed in a French press at 14,0000 lb/in^(t).Lysates were subjected to ultracentrifugation at 40,000×g for 30 min andthe supernatant was subjected to Ni-NTA chromatography, washed withcolumn buffer containing 25 μM imidazole, followed by elution with 500μM imidazole. Eluate was dialyzed with 1×PBS. To remove endotoxin,1:1,000 Triton-X114 was added and the solution was chilled for 5 min,incubated at 37° C. for 10 min, and centrifuged at 13,000×g. Supernatantwas loaded onto a HiTrap desalting column to remove any remnant ofTriton-X114.

TABLE 10 Primers used in this study Primer name Sequence attB1_CoaGGGGACAAGTTTGTACAAAAAAGCAGGCTgatgactaagttgaaaaaagaag (SEQ ID NO: 46)Coa1_BamHI aaGGATCCcctccaaaatgtaattgccc (SEQ ID NO: 47) Coa2_BamHIaaGGATCCgtttgtaactctatccaaagac (SEQ ID NO: 48) attbB2_CoaGGGGACCACTTTGTACAAGAAAGCTGGGTgacacctattgcacgattcg (SEQ ID NO: 49)attB1_vWFGGGGACAAGTTTGTACAAAAAAGCAGGCTcagatagcgattcagattcag (SEQ ID NO: 50)vWF1_BamHI aaGGATCCctgtattttctccttaattttcc (SEQ ID NO: 51) vWF2_BamHIaaGGATCCcatggctgcaaagcaaataatg (SEQ ID NO: 52) attbB2_vWFGGGGACCACTTTGTACAAGAAAGCTGGGTgccctggtgtaacaaatttatg (SEQ ID NO: 53)Coa_promoter_BamHI_FgaaGGATCCgtttattctagttaatatatagttaatg (SEQ ID NO: 54) Coa_out_PstI_RgaaCTGCAGctgtatgtctttggatagagttac (SEQ ID NO: 55) vWbp_promoter_BamHI_FgaaGGATCCggtggcttttttacttggattttc (SEQ ID NO: 56) vWbp_out_PstI_RgaaCTGCAGcgacaaactcattatttgctttgc (SEQ ID NO: 57) Coa_foward_XhoIGAACTCGAGTCTAGCTTATTTACATGG (SEQ ID NO: 58) Coa_Xho_factorXa_FGAACTCGAGatagaaggcagaatagtaacaaaggattatagtggg (SEQ ID NO: 59)Coa_reverse_BamHI GTAGGATCCTGGGATAGAGTTACAAAC (SEQ ID NO: 60)vWbp_forward_XhoI GAACTCGAGgcattatgtgtatcacaaatttggg (SEQ ID NO: 61)vWbp_Xho_factorXa_FGAACTCGAGatagaaggcagagtggtttctggggagaagaatc (SEQ ID NO: 62)vWbp_reverse_BamHI GAACTCGAGgcagccatgcattaattatttgcc (SEQ ID NO: 63)

For enzymatic studies, ELISA, and SPR, full-length coding sequence ofmature Coa or vWbp was cloned into pET15b with primers Coa_Xho_factorXa_F, Coa_reverse_BamHI, vWbp_Xho_factor Xa_F, vWbp_reverse_BamHI (Table10) which contain a Factor Xa site preceding the initial Ile-Val-Thr-Lysof coagulase and Val-Val-Ser-Gly of vWbp. These proteins were expressedand purified using the above protocol, then cleaved with 10 units FactorXa/1 ml for 1 hour at 25° C. to remove the His₆ tag from the N-terminus.Proteins were then loaded onto a Superdex 75 (GE Healthcare) column forfinal purification. All eluted proteins were stored in 1×PBS.

Rabbit antibodies. Protein concentration was determined using a BCA kit(Thermo Scientific). Purity was verified by SDS page gel analysis andCoomassie Brilliant Blue staining Six month old New-Zealand white femalerabbits (Charles River Laboratories) were immunized with 500 μg proteinemulsified in CFA (Difco) for initial immunization or IFA for boosterimmunizations on day 24 and 48. On day 60, rabbits were bled and serumrecovered for immunoblotting or passive transfer experiments. Forantibody purification, recombinant His₆-Coa or His₆-vWbp (5 mg) wascovalently linked to HiTrap NHS-activated HP columns (GE Healthcare).This antigen-matrix was then used for affinity chromatography of 10-20ml of rabbit serum at 4° C. Charged matrix was washed with 50 columnvolumes of PBS, antibodies eluted with elution buffer (1 M glycine pH2.5, 0.5 M NaCl) and immediately neutralized with 1M Tris-HCl, pH 8.5.Purified antibodies were dialyzed overnight against PBS at 4° C.

Surface Plasmon Resonance. Affinity and rates of association anddissociation were measured on a BIAcore 3000. Buffers were sterilefiltered and degassed. A CM5 chip was prepared for amine linkage byinjection of human prothrombin (500 nM, pH 4.0) (Innovative Research)and human fibrinogen (200 nM, pH 4.5) (Innovative Research) in presenceof 0.2 M EDC and 0.05 M NHS. To measure the interaction of coagulasewith prothrombin and fibrinogen, Coa was diluted into HBS-P buffer (20mM HEPES [pH 7.4], 150 mM NaCl, 0.005% [vol/vol] surfactant P20) atconcentrations 0-75 nM with successive injections of coagulase for 300seconds followed by 300 seconds for dissociation followed byregeneration with NaOH (50 μL, 30 seconds). K_(D) and χ² were determinedusing the BiaEvaluation software and best fit was determined with a 1:1binding model with drifting baseline and local R_(max). The interactionof von Willebrand factor with prothrombin and fibrinogen was measured inthe same way. All experiments were repeated in triplicate. Inhibitionexperiments with polyclonal antibodies were conducted by successiveinjections of coagulase (25 nM) incubated with aCoa at 0 nM-200 nM underthe same injection conditions described above. vWF (50 nM) was similarlyincubated with avWF at 0 nM-400 nM. Response difference was measured asthe change in response units from before the injection to the end of theinjection.

Measurements of coagulase activity. 1×10⁻¹⁶ M prothrombin (InnovativeResearch) was pre-incubated for 20 min with an equimolar amount offunctional coagulase or vWbp at room temperature, followed by additionof S-2238 (a chromogenic substrate) to an end concentration of 1 mM in atotal reaction buffer of 100 μl 1×PBS. The change in absorbance wasmeasured at 450 nm for 10 minutes in a spectrophotometer, plotted as afunction of time and fitted to a linear curve. The slope of the curve(dA/dt) was interpreted to be the rate of S-2238 hydrolysis, and thusreflective of enzymatic function (% coagulase-prothrombin orvWbp-prothrombin complex activity). The assay was repeated in presenceof specific or cross antibodies added in 3M excess (3×10⁻¹⁶M) and thedata was normalized to the % average activity without inhibition.

Renal abscess model and lethal challenge. Overnight cultures ofstaphylococcal strains were diluted 1:100 into fresh TSB and grown untilthey reached an OD₆₀₀ of 0.4. 10 ml of bacteria were centrifuged at7,500×g, washed, and suspended in 10 ml of 1×PBS. Six week old femaleBALB/c mice (Charles River) were injected retro-orbitally with 1×10⁷ CFUstaphylococcal suspension in 100 μl of PBS. Cohorts of 10 mice wereused. On the fifth day post infection, these mice were killed by CO₂asphyxiation and their kidneys were excised. All organs were examinedfor surface lesions and 8-10 right kidneys were sent for histopathologysectioning and hematoxylin-eosin staining These slides were examined bylight microscopy for internal abscesses. For the lethal challenge model,all experimental conditions remain the same except that 1×10⁸ CFUstaphylococci were administered and that the mice were monitored for 10days post infection for survival.

Immunohistochemistry staining of renal sections. Sectioned kidneys weredeparafinized and rehydrated through xylene and serial dilutions of EtOHto distilled water. They were incubated in antigen retrieval buffer(DAKO, pH 6.0) and heated in steamer at over 96° C. for 20 minutes.After rinsing, the slides were incubated in 3% hydrogen peroxide for 5minutes and then 10% normal serum in 0.025% Tritonx-100-PBS for 30minutes. 10% human IgG was used as blocking reagent for 30 minutesincubation (Sigma-Aldrich). Primary antibody was applied on the slidesfor over night incubation at 4° C. degree in a humidity chamber. Theprimary antibodies used were 1:500 rat anti-mouse Prothrombin(Innovative Research), 1:500 rabbit anti-mouse fibrinogen (InnovativeResearch), 1:250 rabbit-anti staphylocoagulase, or 1:250 rabbitanti-staphylococcus vwbp. Following TBS wash, the slides were incubatedwith biotinylated secondary antibody (1:50 dilution of biotinylatedanti-rat IgG, BA-4001 from Vector Laboratories; or 1:200 dilution ofbiotinylated anti-rabbit IgG, BA-1000 from Vector), and then ABCreagents (Vector Laboratories). The antigen-antibody binding wasdetected by DAB substrate chromogen system. The slide were brieflyimmersed in hematoxylin for counterstaining and evaluated under lightmicroscope.

Active immunization. Three week old BALB/c mice were injected with 50 μgprotein each, emulsified in 100 μl CFA. Cohorts of 15 mice were used,with 5 mice reserved for bleeding and antibody titers. Eleven days postvaccination, these mice were boosted with 50 μg protein each, emulsifiedin 100 μl IFA. On day 21, mice were injected with 1×107 CFU ofstaphylococci for the renal abscess model or 1×108 CFU for lethalchallenge. At the time of infection 5 mice were bled to obtain antibodytiters.

Passive transfer of antibodies. Twenty four hours prior to infection,six week old BALB/c mice were injected with purified antibodies againstCoa and/or vWbp at a dose of 5 mg/kg body weight. Cohorts of 10 micewere used. These mice were challenged by retro-orbital injection with1×10⁷ CFU (renal abscess model) or 1×10⁸ CFU staphylococci (lethalbacteremia).

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. An isolated polypeptide comprising a variant Protein A (SpA)comprising (a) at least one amino acid substitution that disrupts Fcbinding and (b) at least a second amino acid substitution that disruptsVH3 binding and (c) an amino acid sequence that is at least 70%identical to the amino acid sequence of SEQ ID NO:2.
 2. The isolatedpolypeptide of claim 1, wherein the variant SpA comprises a variantdomain D segment.
 3. The isolated polypeptide of claim 2, wherein theSpA variant comprises one or more amino acid substitutions at amino acidposition 9 or 10 of SEQ ID NO:2 and comprises an amino acid sequencethat is at least 80% identical to the amino acid sequence of SEQ IDNO:2.
 4. The isolated polypeptide of claim 3, wherein at least one ofthe one or more amino acid substitutions is a lysine residue for aglutamine residue.
 5. The isolated polypeptide of claim 3, furthercomprising an amino acid substitution at least one of amino acidpositions 36 and 37 of SEQ ID NO:2.
 6. The isolated polypeptide of claim5, wherein the amino acid sequence of the domain D comprises an alanineresidue substitution at least one of amino acid positions 36 and 37 ofSEQ ID NO:2.
 7. The isolated polypeptide of claim 2, further comprisingone or more variants of an SpA E domain, A domain, B domain, or C domainsegments.
 8. The isolated polypeptide of claim 2, comprising two or moreD domain segments.
 9. The isolated polypeptide of claim 1, furthercomprising a non-Protein A segment.
 10. The isolated polypeptide ofclaim 9, wherein the non-Protein A segment is a second antigen segment.11. The isolated polypeptide of claim 10, wherein the second antigensegment is a staphylococcal antigen segment.
 12. The isolatedpolypeptide of claim 11, wherein the staphylococcal antigen segment isan Emp, EsxA, EsxB, EsaC, Eap, Ebh, EsaB, Coa, vWbp, vWh, Hla, SdrC,SdrD, SdrE, IsdA, IsdB, IsdC, ClfA, ClfB, and/or SasF segment.
 13. Apeptide composition comprising, in a pharmaceutically acceptablecomposition, a non-toxigenic variant Protein A (SpA) peptide having oneor more mutations that attenuate the binding of the Protein A domain Dto IgG, Fcγ, VH3 F(ab)₂, von Willebrand factor (vWF), and tumor necrosisfactor α receptor 1 (TNFR1), wherein the composition is capable ofstimulating an immune response in a subject in need thereof. 14.-18.(canceled)
 19. The composition of claim 13, further comprising at leasta second staphylococcal antigen.
 20. The composition of claim 19,wherein the second antigen selected from an EsaB, Emp, EsxA, EsxB, EsaC,Eap, Ebh, Coa, vWbp, vWh, Hla, SdrC, SdrD, SdrE, IsdA, IsdB, IsdC, ClfA,ClfB, and/or SasF peptide. 21-23. (canceled)
 24. An immunogeniccomposition comprising an isolated peptide comprising a Protein A (SpA)variant having an amino acid substitution at amino acid positions 9, 10,36, and 37 of SEQ ID NO:2.
 25. The composition of claim 24, furthercomprising at least one other staphylococcal antigen or immunogenicfragment thereof selected from the group consisting of: Emp, EsxA, EsxB,EsaC, Eap, Ebh, EsaB, Coa, vWbp, vWh, Hla, SdrC, SdrD, SdrE, IsdA, IsdB,IsdC, ClfA, ClfB, and SasF. 26.-29. (canceled)
 30. A vaccine comprisingthe isolated polypeptide of claim 1 and a pharmaceutically acceptableexcipient.
 31. (canceled)
 32. A method of preventing or treatingstaphylococcal infection comprising the step of administering thevaccine of claim 30 to a patient in need thereof. 33.-38. (canceled) 39.A method for eliciting an immune response against a staphylococcusbacterium in a subject comprising providing to the subject an effectiveamount of a composition comprising a Protein A (SpA) variant having anamino acid substitution at amino acid positions 9 and 10 of SEQ ID NO:2.40.-51. (canceled)
 52. The method of claim 39, further comprisingadministering to the subject a composition comprising a secondstaphylococcal antigen.
 53. The method of claim 52, wherein the secondstaphylococcal antigen is one or more of Emp, EsxA, EsxB, EsaC, Eap,Ebh, EsaB, Coa, vWbp, vWh, Hla, SdrC, SdrD, SdrE, IsdA, IsdB, IsdC,ClfA, ClfB, and SasF. 54.-108. (canceled)
 109. A method of preventing ortreating staphylococcal infection comprising the step of administeringan immunogenic composition comprising a Staphylococcal coagulase or animmunogenic segment thereof.
 110. The method of claim 109, wherein theStaphylococcal coagulase is a Coa or vWh polypeptide or immunogenicsegment thereof. 111.-117. (canceled)
 118. A method of making apolypeptide of claim 1, comprising obtaining the polypeptide from a hostcell.
 119. The isolated polypeptide of claim 4, wherein the one or moreamino acid substitutions comprise a lysine residue for a glutamineresidue at positions 9 and 10 of SEQ ID NO:2.
 120. The isolatedpolypeptide of claim 6, wherein the amino acid sequence of the domain Dcomprises an alanine residue substitution at amino acid positions 36 and37 of SEQ ID NO:2.
 121. The isolated polypeptide of claim 120, furthercomprising substitutions of a lysine residue for a glutamine residue atpositions 9 and 10 of SEQ ID NO:2.